Nano silver kills the virus that causes blue ear disease (PRRS)
This study aimed to investigate the antiviral effect of nano silver (AgNPs) on porcine reproductive and respiratory syndrome virus (PRRSV) in vitro and to preliminarily analyze its mechanism, so as to provide new ideas for the prevention and control of PRRSV. The safe concentrations of AgNPs for subsequent experiments were selected from 0.1875, 0.375, 0.75, 1.5, 3, 6, and 12 μg/mL by CCK-8 kit, which was used to evaluate the toxicity of nano silver AgNPs to Marc145 cells. The antiviral effect of silver nanoparticles against PRRSV was evaluated by microscopic observation, indirect immunofluorescence assay, determination of virus concentration, and real-time quantitative RT-PCR. The direct inactivation effect of AgNPs against PRRSV was evaluated by indirect immunofluorescence assay and real-time quantitative RT-PCR. The effects of AgNPs on the adhesion and invasion of different multiplicities of infection (MOI) of PRRSV (0.0001 to 0.1) to Marc145 cells were analyzed by real-time quantitative RT-PCR. The effects of AgNPs on PRRSV proliferation were analyzed by real-time quantitative PCR after Marc145 cells treated with AgNPs at the indicated time points were infected with PRRSV for 3, 6, 12, 18, and 24 h. The maximum safe concentration of AgNPs for Marc145 cells was 1.5 μg/mL. There were both certain inhibitory and inactivating effects of 0.375, 0.75, and 1.5 μg/mL AgNPs against PRRSV. The adhesion and invasion of different MOI strains into Marc145 cells were inhibited by AgNPs and the proliferation of PRRSV was inhibited by adding nanosilver at 3, 6, 12, 18 and 24 h. AgNPs had anti-PRRSV ability and this resistance was exerted through direct inactivation of PRRSV and inhibition of viral adhesion, invasion and proliferation.
INTRODUCE
Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease with high mortality caused by porcine reproductive and respiratory syndrome virus (PRRSV). The disease was first reported in the United States in 1987 and has now spread to pig-raising countries and regions around the world. The disease was introduced to China in 1996, causing great losses to the pig industry. The genes of PRRSV are susceptible to mutation and recombination, resulting in variety diversity. The prevalence of PRRSV in China has changed greatly from the classical strain CH-1a to the highly virulent PRRSV strain that emerged in 2006, followed by the NADC30 strain reported in 2008. PRRSV can damage the immune system of infected pigs and is susceptible to mixed infections with other pathogens, seriously affecting pig production. At present, the prevention and control of PRRSV mainly rely on the immunity of vaccines, but the cross-protection ability of the same vaccine strain against different strains is weak, which limits its immune efficacy. Therefore, the development of effective drugs against PRRSV for the prevention and control of PRRS is urgent, in order to make up for the shortcomings of vaccine immunity in the prevention and control of PRRSV. Nanomaterials, as novel materials, have been widely used in antibacterial applications due to their unique properties. Among them, silver nanoparticles (AgNPs) combine the properties of silver and nanomaterials, showing strong antibacterial potential and have been used in drug delivery, diagnostic, anti-cancer and antibacterial applications. An advantage of AgNPs in antibacterial applications is that they do not easily develop drug resistance, have multiple sites of action, and can play a role in many bacterial growth and infection processes in the body. Studies have shown that AgNPs have the ability to inhibit a wide range of viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)*1, human immunodeficiency virus (HIV), herpes simplex virus (HSV), Newcastle disease virus (NDV), and porcine epidemic diarrhea virus (PEDV), and are expected to be developed into highly effective and broad-spectrum antivirals. Currently, there are few reports on the anti-PRRSV ability of AgNPs. In this perspective, this study used Marcl45 cells as an in vitro model to explore whether AgNPs have anti-PRRRSV effects in vitro and to preliminarily explore the mechanism of action of AgNPs against PRRSV, aiming to provide new ideas for the prevention and control of PRRSV.
Materials and Methods
Materials
Strains and cells PRRSV strain JXA1 was isolated and preserved by the Laboratory of Swine Disease Research, Institute of Veterinary Medicine, Guangdong Academy of Agricultural Sciences; Marc145 cell line was preserved by the Laboratory of Swine Disease Research, Institute of Veterinary Medicine, Guangdong Academy of Agricultural Sciences. 1.1.2 Main reagent Nanosilver with a concentration of 30,000 µg/ml, particle size of 5-7 nm, density of 1.01 g/cm2, and pH 8.0 was purchased from Guangdong Shunde Zhengshanchuan Biotechnology Co., Ltd.; fetal bovine serum, trypsin, and 1640 basal medium were purchased from Gibeo; primary antibody against PRRSV N protein for immunofluorescence was purchased from MEDIAN Diagnostics; CCK-8 kit and FITC-conjugated secondary antibody were purchased from Beyotime Biotechnology Co., Ltd.; viral DNA/RNA extraction kit was purchased from Magen; and One Step qRT-PCR SYBR Green kit was purchased from Nanjing Novozyme Biotechnology Co., Ltd.
1.2 Methods
1.2.1 PRRSV culture and concentration determination
Inject PRRSV suspension into Marc145 cell culture plate, place in cell culture incubator for 1 hour, remove supernatant, add maintenance solution and continue culture until lesion reaches 80% or more. The virus solution was diluted 10 times in a gradient, with a total of 8 gradients (10-1, 10-2, 10-3, 10-4, 10-5 10-6, 10-7, 10-8), added to a 96-well plate of Marc145 cells, 10 copies for each gradient, 100 μL/well, incubated in a cell culture incubator for 1 hour, removed the supernatant, added maintenance solution, 200 μL/well, continued to culture until complete injury, calculated the concentration by the Reed-Muench method, expressed as the half-lethal dose (TCID/mL).
1.2.2 Indirect immunofluorescence assay (IFA)
PRRSV-infected Marc145 cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% TritonX-100 after 30 min, blocked with 1% BSA at room temperature after 15 min, added primary antibody against N protein (1:1000 dilution) after 30 min and incubated at 4°C overnight, added secondary antibody (1:100 dilution) and incubated at room temperature for 1 h. Unbound secondary antibodies were washed with PBS and cells were observed and photographed under a fluorescence microscope.
1.2.3 Real-time fluorescence quantitative RT-PCR
Total RNA from PRRSV-infected Marc145 cells was extracted and the expression level of PRRSV N gene was detected by real-time fluorescence quantitative RT-PCR. Primer sequences were designed according to Liu et al.a. Primer sequences were: PRRSVN gene, upstream primer: 5′-AGATCATCGCCCAACAAAAC-3′, downstream primer: 5′-GACACAATTGCCGCTCACTA-3′; internal reference gene Bractin, upstream primer. 5′-TCCCTGGAGA-AGAGCTACGA-3′, downstream primer: 5′-AGCACTGT-GTTGGCGTACAG-3′, all primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
20 L PCR reaction system: 2× buffer 10 pL, enzyme 1L, upstream and downstream primers (10µmol/L) 0.4 pL each, RNA 2 juL, deionized water up to 20 L. PCR reaction program: 50°C 3 min 95 30 sec: 95 C 10 sec. 60 C 30 sec. 45 1- Melting curve: 95 °C 15 sec, 60 °C 60 sec, 95 °C 15 sec, relative expression was analyzed by 2-AMD method.
1.2.4 Toxic effects of AgNPs on Marc145 cells
The AgNP stock solution was diluted to 7 concentrations (12, 6, 3, 1.5, 0.75, 0.375, 0.1875 µg/mL) using 1640 culture medium. 0 µg/mL AgNPs were used as the control group and added to Marc145 cells in 96-well plates in sequence. Each concentration was repeated 4 times. A blank control was set at 100 µL/well. The cells were cultured in a cell culture incubator for 2 h. The supernatant was discarded and the maintenance solution was added at 100 µL/well. The cells were cultured for 48 h. CCK-8 solution was added and incubated for 1 h. The absorbance was measured at 450 nm.
1.2.5-Anti-PRRSV infection effect of AgNPs pretreatment on Mare145 cells
AgNPs at different concentrations (0, 1.5, 0.75, 0.375 µg/mL) at 37°C for 1 h, remove the supernatant, wash with PBS and infect Mare145 cells with PRRSV for 1 h, remove the supernatant, wash with PBS and replace the maintenance medium. Continue to culture for 48 h and then perform indirect immunofluorescence assay, the method is similar to 1.2.2: collect the culture supernatant and freeze-thaw three times to measure the concentration, the method is similar to 1.2.1; collect the culture supernatant to extract total RNA and use real-time fluorescence quantitative RT-PCR to detect the expression level of PRRSVN gene, the method is similar to 1.2.3.
1.2.6 Inactivation effect of AgNPs on PRRSV
AgNPs at different concentrations (0, 0.75, 0.375, 0.1875 µg/mL) were mixed with PRRSV at a multiplicity of infection (MOID) of 0.01, incubated at 37°C for 1 h, then injected into Marc145 cells that had grown into a monolayer and in good condition, and continued to culture for 48 h. h later, an indirect immunofluorescence assay was performed, the method was the same as 1.2.2: the culture was collected and frozen and thawed three times to determine the concentration, the method was the same as 1. 2. 1.
1.2.7 Effect of AgNPs on the adhesion of PRRSV to Marc145 cells
The Marc145 cell plate was pre-cooled at 4°C, 0.75 µg/mL AgNPs were mixed with different PRRSV MOI (0.0001~0.1), and the pre-cooled Marc145 cells were inoculated and incubated at 4°C for 2 h, the supernatant was removed, the plate was washed with PBS, and maintenance solution was added. The plate was placed in a cell culture incubator for further culture for 48 h, the culture was collected to extract total RNA, and the expression level of PRRSVN gene was detected by real-time fluorescence quantitative RT-PCR, the same method as 1. 2. 3.
1.2.8 Effect of AgNPs on PRRSV penetration into Marcl45 cells
Marc145 Cell plates were pre-cooled at 4°C and Marcl45 cells were infected with different PRRSV MOL (0.0001-0.1), incubated at 4°C for 2 h, removed the supernatant, washed the plates with PBS and added maintenance solution containing 0.75 µg/mL AgNPs, then cultured the cells in a cell culture incubator for 3 h. Remove the supernatant, wash the plates with PBS and add maintenance solution for further culture for 48 h. Collect the culture supernatant to extract total RNA and detect the PRRSV N gene expression level by real-time fluorescence quantitative RT-PCR. This method is the same as 1. 2. 3.
1.2. 9 Effect of AgNPs on PRRSV proliferation Marcl45 cells that had grown into monolayers and were in good condition were infected with PRRSV, incubated in a cell culture incubator for 1 hour, the supernatant was removed, the plate was washed with PBS and the maintenance solution was added, and the cells were cultured in a cell culture incubator for further culture. After 3, 6, 12, 18 and 24 hours, the maintenance solution was replaced with 0.75 µg/mL AgNPs and the cells were cultured in a cell culture incubator for further culture. After 48 hours, the culture supernatant was collected to extract total RNA and detect the expression level of PRRSVN gene by real-time fluorescence quantitative RT-PCR, this method is the same as 1.2.3.
1.3 Statistical analysis of data
Experimental data were preliminarily processed by Excel 2016 software and experimental data were subjected to one-way analysis of variance and graphed by GraphPad Prism 8.0 software. The results were expressed as mean ± standard deviation, P < 0.05 indicated a significant difference; P < 0.01 indicated an extremely significant difference.
Results
Toxic effects of AgNPs on Marc145 cells
As shown in the figure, AgNPs have certain toxicity to Marc145 cells. Compared with the 0 µg/mL AgNP group, the cell viability after treatment with 3, 6, and 12 µg/mL AgNPs was significantly reduced (P < 0.01), and the cell viability at 0.1875, 0.375, 0.75, and 1.5 µg/mL silver nanoparticles was significantly reduced. There was no significant change in cell viability after treatment (P > 0.05). Therefore, a safe silver nano concentration below 1.5 µg/mL was selected for further experiments.
Compared with the 0 µg/mL AgNP group, *, the difference is significant (P < 0.05); ***, the difference is extremely significant (P < 0.01). Figures 3 and 5 are similar
Compared to AgNP 0 ng/mL, **Significant difference
(P < 0.05);**, Extremely significant difference (P < 0.01).
Same as Figures 3 and 5
Figure 1 Toxic effects of AgNPs on Marcl45 cells
Effect of AgNP nano silver on PRRSV infection before Marc145 cell treatment
Marc145 cells were pretreated with AgNP nanosilver and then infected with PRRSV virus. The indirect immunofluorescence results are shown in Figure 2. As shown in Figure 2, compared with the 0 µg/mL AgNP group, the degree of cell pathology in the 0.375, 0.75, 1.5 µg/mL AgNP treatment group was weaker and the number of immunofluorescence-positive cells decreased. To further verify the anti-PRRSV infection effect of cells pretreated with AgNPs, infected cultures were collected to detect the PRRSV N gene expression level and determine the PRRSV virus concentration, and the results are shown in Figure 3. As shown in Figure 3, compared with the 0 µg/mL AgNP group, the PRRSV N gene expression levels of cells in the 0.375, 0.75, and 1.5 µg/mL AgNP treatment groups were significantly or extremely significantly reduced (P < 0.05; P < 0.01): the virus concentration of cells in the 0.375 µg/mL nanosilver treatment group had no significant change (P>0.05), and the virus concentration of cells in the 0.75 and 1.5 µg/mL AgNP treatment groups was extremely significantly reduced (P < 0.01). This suggests that Marc145 cells pretreated with AgNPs have some resistance to PRRSV infection,
Figure 2 Indirect immunofluorescence results of nano silver AgNPs against PRRSV in pretreated Marcl45 cells (200X)
Inactivation effect of nano silver AgNPs against PRRSV
After co-incubation with PRRSV, nano silver AgNPs infected Marc145 cells. The results of indirect immunofluorescence are shown in Figure 4. As shown in Figure 4, compared with the 0 µg/mL AgNPs group, the degree of cell damage and the number of immunofluorescence-positive cells in the 0.375, 0.75, 1.5 µg/mL AgNPs groups were reduced. Among them, there were no damaged cells in the 1.5 µg/mL AgNPs treatment group. To further verify the inactivation effect of AgNPs against PRRSV, infected culture samples were collected to determine the virus concentration. The results are shown in Figure 5. As shown in Figure 5, compared with the 0 µg/mL AgNP group, the virus concentrations in the 0.375, 0.75, and 1.5µg/mL AgNP treatment groups were significantly reduced (P < 0.01), and no cytopathic effect was observed in the 1.5µg/mL AgNP treatment group, indicating that AgNPs had a certain inactivating effect on PRRSV.
Figure 4 Indirect immunofluorescence results of the inactivation effect of AgNPs on PRRSV (200X)
Figure 5 Determination of viral titer of inactivation effect of AgNP nano silver against PRRSV inactivation effect
of AgNPs against PRRSV
2.4 Effect of nano silver AgNPs on PRRSV Adhesion to Marc145 Cells
As shown in Figure 6, when cells were infected with PRRSV at 0.0001 MOI, pretreatment with nano silver AgNPs significantly reduced the expression level of PRRSV N Gene in infected cells (P < 0.01); when MOI was increased stepwise and PRRSV MOI of 0.001, 0.01 and 0.1 MOI was used for infection, AgNPs treatment also significantly or significantly reduced the expression level of PRRSV N Gene in infected cells (P < 0.05; P < 0.01).
2.5 Effect of AgNPs on PRRSV invasion into Marc145 cells
As shown in Figure 7, when cells were infected with PRRSV MOI 0.0001, AgNPs treatment significantly reduced the expression level of PRRSV N gene in infected cells (P < 0.01); when MO1 was increased stepwise, cells were infected with 0.001, 0.01 and 0.1 MOI PRRSV, AgNPs treatment significantly or significantly reduced the expression level of PRRSV N gene in infected cells (P < 0.05; P < 0.01),
Figure 6 Effect of AgNPs on PRRSV adhesion to Marc145 cells
Figure 7 Effect of nano silver AgNPs on PRRSV invasion into Mare145 cells
2.6 Effect of nano silver AgNPs on PRRSV Growth
As shown in the figure, the expression level of PRRSV N gene was significantly or extremely significantly reduced (P < 0.05; P < 0.01) when AgNPs were added at different time points (3, 6, 12, 18, 24 h) after cells were infected with PRRSV.
Figure 8 Effect of nano silver AgNPs on PRRSV growth
3 Discussion
Antiviral mechanism of AgNPs. The size, morphology, surface, particle distribution and concentration of AgNPs affect the toxicity of AgNPs. High concentrations of AgNPs have toxic effects on cells and may cause cell death. The premise of antiviral drug development must be non-toxic. Therefore, this experiment first analyzed the toxicity of AgNPs to Marc145 cells and selected a concentration of 1.5ug/mL or less that is non-toxic to cells as the safe concentration for subsequent experiments. PRRSV infection in Marc145 cells can cause obvious lesions. After using AgNPs to pre-treat Marcl45 cells and then infecting them with PRRSV, the degree of lesions was significantly reduced. The infected culture sample was collected for quantitative real-time fluorescence RT-PCR to detect the expression of PRRSVN gene. The results showed that the expression level was significantly or extremely significantly reduced. The determination of the virus concentration also obtained similar results, demonstrating that AgNPs have certain resistance to PRRSV.
Silver nanoparticles have become a research hot spot due to their unique physical and chemical properties and broad antibacterial properties. It has been confirmed that they have certain resistance to bacteria, fungi and viruses. Among them, the antiviral effect has attracted much attention, and relevant studies on AgNPs against SARS-CoV2 have been carried out. PRRS poses a serious threat to the global pig industry. Prevention and control of PRRS are very important for healthy pig farming. The prevention and control of PRRS currently relies on vaccines, but vaccine prevention and control have certain limitations. Therefore, the development of drugs against PRRSV is of great significance. This study conducted in vitro experiments to explore the effect of AgNPs on PRRSV and preliminarily explored the infection process of PRRSV, and the development of antiviral drugs mainly includes direct inactivation of the virus and interference with the infection process of the virus. Yang et al. found that curcumin-modified AgNPs (cAgNPs) could directly inactivate human respiratory syncytial virus (RSV), and the inactivation effect was better than that of citric acid-modified AgNPs. AgNPs had a certain inactivation effect on human norovirus-like viruses, including feline calicivirus (FCV) and murine norovirus (MNV), and AgNPs modified with 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV18) (PHBV18/
AgNPs) had a stronger inactivation effect on the virus. This study analyzed the neutralizing effect of AgNPs on PRRSV. The results showed that AgNPs had a certain neutralizing effect on PRRSV. After PRRSV was treated with AgNPs and then infected into Marc145 cells, the PRRSV virus concentration was significantly reduced. TA-modified AgNPs (TA-AgNPs) can exert anti-HSV-2 effects by inhibiting viral adhesion and invasion into cells. AgNPs with a particle size of 10 nm can effectively inhibit SARS-CoV-2 in vitro. The anti-SARS-CoV2 mechanism is mainly achieved by inhibiting viral invasion into cells. This study found that AgNPs can inhibit PRRSV adhesion and invasion into Marc145 cells and have a certain inhibitory effect on PRRSV infection at different MOIs. Du et al.31 found that silver sulfide nanoparticles (Ag:S nanoclusters, Ag2S NC) could inhibit the growth of PEDV. Compared with the control group, the PEDV virus concentration could be reduced by 3 orders of magnitude after 12 h of exposure to AgzS NC. After entering the cells, PRRSV multiplied and grew, producing more virus particles and expanding the infection. Therefore, this study further analyzed the effects of silver nanoparticles AgNPs on the growth of PRRSV after entering Marc145 cells.
The results showed that AgNPs had a certain inhibitory effect on the growth of PRRSV after entering Marc145 cells, and the addition of AgNPs at different times showed an inhibitory effect. This study has preliminarily explored the mechanism of action of AgNPs against PRRSV, but its specific antiviral mechanism still needs to be further studied.
4 Conclusions
This study determined from the in vitro level that AgNPs with a concentration lower than 1.5 µg/mL had no effect on Marc145 cells, which is a safe concentration, and AgNPs had an anti-PRRSV effect within the safe concentration range, further confirming that AgNPs have an anti-PRRSV effect through multi-target effects, not only directly inactivating PRRSV, but also having a certain inhibitory effect on the adhesion, invasion, and growth of PRRSV during infection.
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