Application of nano-zinc oxide in the preparation of drugs against fish iridovirus

Abstract

This invention discloses the application of nano-zinc oxide in the preparation of drugs against fish iridovirus (SGIV). The study shows that nano-zinc oxide (ZnONPs) can significantly attenuate the cytopathic effect induced by SGIV infection, reducing the mRNA transcription level, protein expression level, and viral titer of the major SGIV capsid protein. Treatment with ZnONPs can also enhance the host's interferon immune-related genes ISG56 and ISG20, and anti-inflammatory factors IL-10 and TGF-β. β The ZnONPs significantly reduced the mRNA transcription level, thereby decreasing the infectivity of SGIV to cells and demonstrating antiviral activity against fish iridoviruses. Furthermore, ZnONPs exhibited low cytotoxicity and good safety profile, making them suitable for the preparation of drugs to prevent and treat grouper iridovirus disease. This invention provides a novel drug intervention strategy for the prevention and treatment of SGIV infection and has significant application value in the control of iridovirus-related diseases in aquaculture.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and aquaculture technology, and more specifically, to the application of nano zinc oxide in the preparation of drugs against fish iridovirus. Background Technology

[0002] Grouper belongs to the order Perciformes, family Serranidae, genus Grouper. Epinephelus The wrasse (also known as the common wrasse) gets its name from its preference for inhabiting rocky areas and the patterns and spots covering its body. As a warm-water fish, it is widely distributed in tropical and subtropical seas. Rich in various nutrients and with delicious, flavorful flesh, it is highly popular among consumers. Furthermore, due to its rapid growth and reproduction, strong resilience, and high economic value, it is favored in aquaculture. Currently, diversified aquaculture models have been developed, including pond culture, cage culture, and factory farming, covering coastal provinces such as Guangdong, Fujian, Hainan, and Guangxi, and it occupies an important position in my country's marine aquaculture industry.

[0003] In recent years, with the continuous expansion of grouper farming, the degradation of farmed species and other factors have led to frequent outbreaks of diseases. Among these, Singapore grouper iridovirus (SGIV) is one of the common pathogens in grouper farming. Fish infected with this virus exhibit symptoms such as darkening of body color, decreased appetite, and swollen, darkened spleen, with a mortality rate as high as 90%. SGIV belongs to the genus Ranavirus in the family Iridoviridae. Its genome size is 140,131 bp, encoding 162 open reading frames (ORFs). The encoded proteins include not only structural proteins such as the major capsid protein MCP and envelope proteins VP019 and VP088, but also some enzymes essential for viral genome replication and immune escape proteins. Due to the current lack of effective control measures, SGIV infection has caused significant economic losses to grouper farming in my country, becoming a major bottleneck in the industry. Therefore, it is urgent to develop new control strategies to ensure the healthy and sustainable development of the industry.

[0004] Zinc, an essential trace element for animals, is known as the "element of life" due to its wide range of physiological and biochemical functions in the body. Nano-zinc oxide, with its small particle size, can penetrate interstitial spaces, thus exhibiting more efficient biological activity. For example, adding nano-zinc oxide to animal feed can improve the efficiency of feed absorption and utilization; simultaneously, nano-zinc oxide added to feed can enhance the antioxidant properties of animals after absorption. Currently, the application of nano-zinc oxide in aquaculture mainly focuses on its effects on the growth performance of aquatic animals as a feed additive; there are no reports on its application in the treatment of diseases caused by fish viruses such as iridovirus infection. Therefore, this invention application is filed. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of existing SGIV treatment drugs. This invention provides the application of nano zinc oxide in the preparation of drugs against fish iridovirus.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides the first study demonstrating that nano-zinc oxide (ZnONPs) possesses antiviral activity against fish iridovirus SGIV. Cellular experiments showed that nano-zinc oxide significantly attenuated the cytopathic effect (CPE) induced by SGIV infection. Quantitative real-time PCR (qPCR) results revealed that nano-zinc oxide significantly downregulated the mRNA transcription levels of key SGIV structural protein genes (MCP, VPO19). Western blot analysis showed that nano-zinc oxide effectively inhibited the expression level of the major capsid protein MCP. Virus titer experiments showed that nano-zinc oxide significantly reduced the viral titer. Therefore, nano-zinc oxide treatment can significantly reduce the pathogenicity of SGIV to cells, exhibiting antiviral activity against fish iridovirus SGIV, with low cytotoxicity and good safety, making it suitable for the preparation of drugs for the prevention and treatment of grouper iridovirus disease.

[0007] Therefore, the present invention provides the following applications of nano zinc oxide: Application in the preparation of drugs against fish iridovirus SGIV.

[0008] Preferably, the fish is a grouper.

[0009] Application in the preparation of drugs for the prevention and treatment of grouper iridovirus disease.

[0010] Application in the preparation of products that reduce or alleviate grouper iridovirus disease.

[0011] Preferably, the product is food, medicine, feed additive, etc.

[0012] Preferably, the drug or product can reduce the cytopathic effect (CPE) caused by SGIV infection.

[0013] Preferably, the drug or product can reduce the expression levels of key SGIV genes (MCP, VP019) and the major capsid protein MCP.

[0014] Preferably, the drug or product can significantly reduce viral titers and weaken the pathogenicity of SGIV to cells.

[0015] Specifically, the nano-zinc oxide provided by this invention is biosynthesized. Based on the research of this invention, it is foreseeable that nano-zinc oxide prepared from other sources can achieve the same effects, or commercially available nano-zinc oxide can be used directly with the same results. Biosynthesized nano-zinc oxide has good environmental friendliness and biocompatibility, requires no toxic reagents for preparation, conforms to the concept of green chemistry, and its plant-derived biomolecules encapsulate ZnONPs, exhibiting low cytotoxicity and suitability for pharmaceutical and food applications. Its activity and stability are better than ZnONPs prepared by chemical / physical methods.

[0016] Preferably, the nano zinc oxide is biosynthesized nano zinc oxide, prepared by Bacillus creboni.

[0017] More preferably, the biosynthesized nano zinc oxide is produced by Bacillus creboni (… Paenibacillus kribbensis The aerospace mutant strain △PS04-17 was synthesized and prepared.

[0018] More preferably, the *Bacillus cristatus* (Cribenzia) Paenibacillus kribbensis The space-adapted mutant strain △PS04-17 was deposited on March 17, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No:62233, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0019] Furthermore, the preparation method of the biosynthesized nano zinc oxide is as follows: after activating the Cribben-type Bacillus aerospace mutant strain △PS04-17, it is inoculated into a fermentation medium containing zinc nitrate hexahydrate for fermentation culture. The fermentation broth is centrifuged, the precipitate is collected, washed, centrifuged again, purified, and freeze-dried to obtain △PS04-17 biosynthesized nano zinc oxide.

[0020] Furthermore, the drug can alleviate cytopathic effects and inhibit the transcriptional expression of key SGIV genes.

[0021] Furthermore, the key genes of the iridovirus SGIV are the major capsid protein MCP and the envelope protein VP019.

[0022] Preferably, the concentration of nano zinc oxide in the drug or product is 3–50 μg / mL.

[0023] Furthermore, the concentration of nano zinc oxide in the drug is 3.125–12.5 μg / mL.

[0024] Furthermore, the concentration of nano zinc oxide in the drug is 6.25–12.5 μg / mL.

[0025] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0026] More preferably, the excipients are selected from pharmaceutically acceptable carriers and excipients.

[0027] Preferably, the drug is prepared as a powder, capsule, or granule.

[0028] The present invention has the following beneficial effects: This invention provides a novel application of nano-zinc oxide in the preparation of drugs against iridovirus. Studies show that nano-zinc oxide can significantly reduce the cytopathic effect (CPE) induced by SGIV infection, significantly downregulate the mRNA transcription levels of key SGIV structural protein genes (MCP, VPO19), effectively inhibit the expression level of the major capsid protein MCP, and significantly reduce viral titer. Therefore, nano-zinc oxide treatment can significantly reduce the pathogenicity of SGIV to cells, exhibiting an anti-SGIV effect with low cytotoxicity and good safety, and can be used to prepare drugs for the prevention and treatment of iridovirus disease in grouper. This invention provides a new drug intervention strategy for the prevention and treatment of SGIV infection and has important application value for the prevention and control of SGIV-related diseases in aquaculture. Attached Figure Description

[0029] Figure 1 The results of detecting the viability of GS cells treated with different concentrations of nano zinc oxide using the CCK-8 assay are shown in the figure (A in the figure represents the statistical results of cell viability; B represents the results of cell state).

[0030] Figure 2 The figure shows the results of viral infection of CPE after GS cells were treated with different concentrations of nano zinc oxide.

[0031] Figure 3 The results of viral gene transcription levels after treating GS cells with different concentrations of nano zinc oxide are shown in the figure (A in the figure is the relative expression level of SGIV MCP gene; B is the relative expression level of SGIV VP19 gene).

[0032] Figure 4 The figure shows the effects of different treatments of nano-zinc oxide on viral gene transcription regulation.

[0033] Figure 5 Figure shows the results of detecting the expression level of viral MCP protein in GS cells infected with SGIV after treatment with different concentrations of nano zinc oxide.

[0034] Figure 6 The figure shows the results of detecting viral titers in GS cells treated with different concentrations of nano zinc oxide after infection with SGIV.

[0035] Figure 7The results of detecting the transcriptional expression of interferon-stimulated genes in GS cells treated with nano-zinc oxide after infection with SGIV are shown in the figure (A in the figure is the relative expression level of IL-10 gene; B is the relative expression level of TGF-B gene).

[0036] Figure 8 Figure 1 shows the transcriptional levels of anti-inflammatory factors in GS cells treated with nano-zinc oxide after infection with SGIV (A represents the relative expression level of the ISG56 gene; B represents the relative expression level of the ISG20 gene). Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] Example 1: Preparation and Cytotoxicity Experiment of Nano Zinc Oxide 1. Preparation of nano zinc oxide The preparation of nano zinc oxide was carried out according to the method described in Chinese patent (patent application number: CN202511390819.8), and the specific operation was as follows: The Cribbenzia space mutant strain △PS04-17 (using Cribbenzia...) was used. Paenibacillus kribbensisThe aerospace mutant strain △PS04-17 was deposited on March 17, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number: GDMCC No: 62233, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. After activation, it was inoculated into a fermentation medium containing zinc nitrate hexahydrate (zinc nitrate hexahydrate 1–5 g / L, sucrose 25–30 g / L, dipotassium hydrogen phosphate 1–1.3 g / L, magnesium sulfate heptahydrate 1–1.2 g / L, potassium chloride 0.5–0.8 g / L, ferrous sulfate heptahydrate 0.01–0.03 g / L, pH 6.5–7.5) for fermentation. The fermentation conditions were 28℃ and static incubation for 10 days. The fermentation broth was centrifuged at 10,000 rpm for 8 min, the precipitate was collected, and washed twice with sterile water, then twice with 0.9% NaCl solution. Sterile water was added to adjust the cell volume of the precipitate, and the mixture was sonicated on ice. The sonication conditions were set to 450 W power, 5-second run followed by a 5-second pause, for a total run time of 30–40 min. After sonication, the mixture was centrifuged at 12,000 rpm for 10 min, and the precipitate was washed three times consecutively with 1.5 mol / L Tris-HCl buffer (pH 8.3). Centrifugation yielded ΔPS04-17 biosynthesized nano-zinc oxide precipitate. The precipitate was then resuspended in ultrapure water, 2 mL of n-octanol was added, and the mixture was shaken for 5 min, centrifuged at 3000 rpm for 5 min, and placed in a 4°C refrigerator for 24 h to allow for clear phase separation. Once the zinc oxide nanosheets precipitated at the bottom of the centrifuge tube, the cells between the two phases were discarded, and the precipitate was washed twice with sterile water and dried in a supercritical dryer. After drying for approximately 1 hour, pure nano-zinc oxide (ZnONPs) was obtained.

[0040] 2. Drug toxicity test of nano zinc oxide Grouper spleen (GS) cells were passaged and seeded at 100 μL / well in 96-well plates and cultured overnight at 28°C in Leibovitz's L-15 complete medium containing 10% fetal bovine serum (FBS). The prepared nano-zinc oxide was then serially diluted with L-15 medium containing 10% FBS to final concentrations of 0, 3.125, 6.25, 12.5, 25, and 50 μg / mL, 100 μL per well, with six replicates per group. After 48 h of GS cell culture, 10 μL of CCK-8 solution was added to each well, gently mixed, and incubated at 28°C in the dark for 1–4 h. The absorbance (OD value) of each well was measured using a multi-mode microplate reader at 450 nm. The relative cell viability (%) was calculated as: (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0041] The results are as follows Figure 1 As shown in Figure A, compared with the control group, cells treated with zinc oxide nanoparticles at concentrations ranging from 3.125 to 12.5 μg / mL maintained cell viability above 90% for 48 h, while the cell viability of the 25–50 μg / mL concentration groups was significantly different from that of the control group. Meanwhile, there was no significant difference in cell state and morphology between the 6.125 and 12.5 μg / mL zinc oxide nanoparticle treatment groups and the untreated group (e.g., ...). Figure 1 (As shown in B in the figure). The above results indicate that 6.125 and 12.5 μg / mL of nano zinc oxide have no significant toxicity to GS cells and can be used for subsequent virus infection experiments.

[0042] Example 2: Detection of Pathogenic Severity and Gene Transcription of Antiviral Activity of Nano-Zinc Oxide 1. Preparation and preservation of SGIV Grouper spleen (GS) cells were inoculated into 25 cm... 2 Cells were cultured in flasks at 28°C for 18 h. SGIV virus (isolated and stored in our laboratory) was added at a multiplicity of infection (MOI) of 1, and the cells were cultured for another 48–72 h. When more than 90% of the cells became rounded and CPE appeared, the cell culture was collected, frozen at -20°C for 3 h, and the freeze-thaw cycle was repeated three times. The cells were then centrifuged at 12000×g at 4°C for 5 min, and the supernatant was collected, aliquoted, and stored at -80°C for long-term use as the viral solution for subsequent experiments.

[0043] Virus identification: Identification was performed using PCR sequencing and TCID. 50 Methods such as assays are used to identify the virus's molecular biology and biological activity, ensuring that the virus titer and purity meet experimental requirements.

[0044] 2. Effects of nano-zinc oxide pretreatment on the severity of viral infection and viral gene transcription GS cells in logarithmic growth phase were trypsinized and seeded into 24-well cell culture plates, and cultured at 28°C until a monolayer of adherent cells formed. The original culture medium was discarded, and culture medium containing different concentrations (6.125 and 12.5 μg / mL) of nano-zinc oxide was added. A control group (MOCK) was also set up, with three replicates per group. After 12 hours of pretreatment, SGIV virus solution prepared in Example 1 was inoculated at MOI=2. 24 hours after infection, the CPE characteristics of each group of cells were observed and recorded using an inverted optical microscope. Simultaneously, according to the Ce1l Total RNA Isolation Kit (FOREGENE, product number RE-03113), cells from different groups were collected, and total RNA was extracted from the cells. cDNA synthesis was performed on the obtained total RNA using the reverse transcription system (10 μL reaction volume) shown in Table 1 below; further, the viral gene transcription level was detected using the qPCR reaction system shown in Table 2 on an Applied Biosystems QuantStudio 5 real-time PCR instrument. Detailed steps of RNA extraction, reverse transcription, and real-time PCR detection are as follows: (1) Cell Total RNA Isolation Kit Operation Procedure: a. Add 250 μL of Buffer cRL1 lysis buffer to the collected cell pellet. After complete lysis, transfer the lysis buffer to the DNA-Cleaning Column, centrifuge at 12000 rpm for 2 min, and collect the supernatant. b. Add 1.6 times the volume of Buffer cRL2 (approximately 400 μL) to the obtained supernatant and mix well; then transfer to RNA-only column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; c. Add 500 μL of Buffer RW1 to the purification column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; add 700 μL of Buffer RW2 again, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. d. Repeat the Buffer RW2 washing step once; centrifuge the empty column at 12000 rpm for 2 min; e. Transfer the purification column to a new 1.5 mL RNase-free EP tube, add 30 μL of RNase-Free ddH2O preheated at 65℃, and let stand at room temperature for 2 minutes; f. Centrifuge at 12000 rpm for 1 minute to collect the RNA solution, and take 1 μL to determine the RNA concentration and purity using Nanodrop.

[0045] (2) The RNA reverse transcription procedure is as follows: a. Heat the extracted RNA at 65°C for 5 min, then immediately place it on ice for 5 min.

[0046] b. Using ReverTraAce ® Prepare a 10 μL reverse transcription reaction system according to Table 1 using the qPCR RT Kit (TOYOBO, product number FSQ-101B). Then perform reverse transcription at 37°C for 15 min, followed by 98°C for 5 min, and finally 16°C for 5 min. The reverse transcription product can be used immediately or stored at -20°C for long-term storage.

[0047] Table 1 Reverse transcription system

[0048] (3) The qPCR experimental procedure is as follows: Prepare a 10 μL reaction mixture using 2×SYBR Green Real-time PCR Master Mix according to Table 2, with four replicate wells for each sample. The reaction was performed on an Applied Biosystems QuantStudio 5 real-time PCR instrument under the following conditions: 95℃ for 1 min; (95℃ for 5 s; 60℃ for 10 s; 72℃ for 15 s) × 40 (40 cycles). β - Actin ( β -actin) was used as an internal reference gene, according to 2 -ΔΔCt The method calculates the relative expression level of the target gene.

[0049] Table 2. Fluorescence Quantitative Analysis System

[0050] The results of cytopathic effect observation showed that, compared with the control group, the number of GS cells pretreated with different concentrations of nano zinc oxide spherically decreased 24 h after SGIV (MOI=2) infection, indicating that the severity of CPE in the nano zinc oxide treatment group was significantly weaker than that in the control group. These results suggest that nano zinc oxide may have the ability to inhibit SGIV infection. Figure 2 qPCR results showed that, compared with the control group, the transcription levels of viral genes (MCP and VP19) in infected cells treated with different concentrations of zinc oxide nanoparticles were significantly decreased. Figure 3 This indicates that pretreatment with nano-zinc oxide can inhibit the transcription of SGIV virus genes.

[0051] 3. Effects of different treatments with nano-zinc oxide on viral gene transcription GS cells in logarithmic growth phase were trypsinized and seeded into 24-well cell culture plates, and cultured at 28°C until a monolayer of adherent cells formed. Four groups were treated with nano-zinc oxide: the pretreatment group (pre group) was treated with 12.5 μg / mL nano-zinc oxide for 12 h, followed by inoculation with SGIV virus at an MOI of 2; the simultaneous treatment group (Co group) was treated with a mixture of 12.5 μg / mL nano-zinc oxide and virus (MOI=2) before inoculation into GS cells; the posttreatment group (Post group) was infected with SGIV virus at an MOI of 2 for 4 h, followed by the addition of 12.5 μg / mL nano-zinc oxide. The control group (MOCK group) received no nano-zinc oxide and was inoculated with SGIV virus at the same time as the other groups. For all groups, virus-infected cells were collected 24 h after SGIV infection, total RNA was extracted, reverse transcribed, and then qPCR was performed to detect the MCP transcription level of the viral gene.

[0052] The results are as follows Figure 4 As shown, compared with the control group, GS cells pretreated with nano-zinc oxide (pre group) exhibited the most significant transcriptional inhibition of the MCP gene 24 h after SGIV (MOI=2) infection. The transcriptional inhibition of MCP in the groups with simultaneous addition of nano-zinc oxide and virus (Co group) and the nano-zinc oxide post-treatment group (Post group) was relatively weaker than in the pre-treatment group, but still showed significant inhibition compared to the control group (MOCK group). This indicates that nano-zinc oxide also has the ability to inhibit viral replication after SGIV infection.

[0053] Example 3: Viral protein expression analysis of antiviral activity of nano-zinc oxide GS cells in logarithmic growth phase were trypsinized and seeded into 6-well cell culture plates, and cultured at 28°C until a monolayer of adherent cells formed. The original culture medium was discarded, and culture medium containing different concentrations (6.125 and 12.5 μg / mL) of nano-zinc oxide was added. A control group (MOCK) was also set up. After 12 h of pretreatment, SGIV virus solution prepared in Example 1 was inoculated at an MOI of 2. Cell pellets were collected 24 h after infection for Western blotting analysis.

[0054] The specific procedures for Western blotting of proteins are as follows: (1) Sample preparation: Add 40 μL Pierce IP lysis buffer to the collected cell pellet and lyse for 30 min; add 10 μL 5× protein loading buffer and mix well; boil in a water bath at 100℃ for 5 min to denature the protein, then centrifuge the sample at 12000×g for 5 min, collect the supernatant and store it at -20℃ for later use. (2) Preparation of protein gels: 10% separating gel and 5% stacking gel were prepared using the SDS-PAGE gel preparation kit (KeyGen BioTECH); (3) Electrophoresis: The prepared gel was loaded into the electrophoresis tank, 1×SDS-PAGE electrophoresis buffer was added, and 10 μL of the prepared protein sample was loaded into each well. Then, the electrophoresis was performed at low voltage of 60 V for 30 min, followed by high voltage of 120 V for 60 min. (4) Transfer: After electrophoresis, remove the protein electrophoresis gel and soak the PVDF membrane (Milipore) in methanol for 30 seconds. Then assemble the transfer clip in the order of "sponge-filter paper-gel-membrane-filter paper-sponge" and transfer at a constant current of 100 mA for 60 min. (5) Blocking: After the transfer, block the membrane with PBST solution containing 5% skim milk in a shaker at room temperature for 2 hours; (6) Antibody incubation: Dilute the primary antibody (MCP, 1:3000 dilution; Tubulin, 1:5000 dilution) according to the ratio, incubate on a horizontal shaker for 2 hours or incubate overnight in a refrigerator at 4°C; then wash the membrane twice with PBST for 5 min each time, and put the membrane into HRP-labeled secondary antibody (1:5000) and incubate at room temperature for 45 min; (7) Color development and photographic analysis: After the secondary antibody incubation, the membrane was washed 3 times with PBST for 10 min each time; chemiluminescent solution was used for color development, and the signal was collected and photographed using a chemiluminescent imaging system.

[0055] The results are as follows Figure 5 As shown, compared with the control group, the expression of viral MCP in the nano zinc oxide treatment group was significantly reduced, indicating that nano zinc oxide treatment can inhibit the expression of viral structural protein MCP.

[0056] Example 4: Titer determination and analysis of the antiviral activity of nano zinc oxide The viral titer in the cells collected from the nano zinc oxide treatment group in Example 3 was determined, and the specific operating steps are as follows: (1) Trypsin digestion was performed on GS cells in the logarithmic growth phase in the culture flask to obtain a cell suspension which was then seeded into a 96-well plate (100 μL / well). (2) After the GS cells grew and formed a monolayer, the virus solution of the control group and the nano zinc oxide treatment group was serially diluted tenfold with a culture medium containing 10% serum (10... -1 ~10 -10 ); (3) Each dilution was inoculated into 8 wells (100 μL / well) for replication, and an uninoculated control was set up at the same time; (4) Observe the occurrence of cytopathic effects (CPE) daily and calculate TCID using the Reed-Muench method. 50 .

[0057] The results are as follows Figure 6 As shown, consistent with the Western blot results, the SGIV titer in cells treated with nano zinc oxide showed a significant decreasing trend compared with the control group, indicating that nano zinc oxide treatment can significantly reduce the generation of progeny viruses.

[0058] Example 5: Host disease resistance gene transcription detection and analysis of antiviral activity of nano-zinc oxide The specific steps for evaluating the regulatory effects of nano-zinc oxide treatment on anti-inflammatory factors and interferon-stimulated gene transcription in host cells after SGIV infection are as follows.

[0059] (1) The GS cell suspension after trypsin digestion was seeded into a 24-well plate and cultured at 28°C until a monolayer of adherent cells was formed.

[0060] (2) The cells were divided into four groups: the first group was the control group without the addition of nano zinc oxide treatment and without SGIV; the second group was the experimental group with the addition of nano zinc oxide treatment but without SGIV; the third group was the experimental group without the addition of nano zinc oxide treatment but with SGIV; and the fourth group was the experimental group with the addition of nano zinc oxide treatment and with SGIV.

[0061] (3) The specific method of nano-zinc oxide treatment is as follows: After the GS cells in the 24-well plate grow into a monolayer of adherent cells, the original culture medium of the second and fourth groups is discarded, and culture medium containing 12.5 μg / mL ZnONPs is added respectively; at the same time, the original culture medium of the first and third groups is discarded, and normal L15 culture medium is added respectively. After 12 h of nano-zinc oxide pretreatment, the third and fourth groups are inoculated with the SGIV virus solution prepared in Example 1 at MOI=2.

[0062] (4) Cell samples from different groups were collected 24 h after SGIV virus infection, and total RNA was extracted from the cells. After obtaining the total RNA, cDNA synthesis was performed using a reverse transcription system (10 μL reaction volume) and a specific program. The results were then analyzed using a quantitative real-time reaction system and a quantitative real-time amplification program in GS cells. IL-10 , TGF-β , ISG56 , ISG20 The relative expression level of genes.

[0063] qPCR results showed that, in the absence of viral infection, treatment with nano-zinc oxide upregulated anti-inflammatory factors in host cells, including... IL-10 and TGF-β The mRNA transcription level was reduced, and the inhibitory effect of SGIV infection on the transcription of the aforementioned anti-inflammatory factor mRNAs was weakened. Figure 7 Similarly, nano-zinc oxide treatment can upregulate the interferon-stimulated gene in host cells. ISG56 and ISG20 The mRNA transcription level, and the inhibitory effect of SGIV infection on the above-mentioned interferon-stimulated gene mRNA transcription ( Figure 8 This indicates that treatment with nano-zinc oxide can enhance interferon and inflammatory responses in host cells.

[0064] The results in summary indicate that nano-zinc oxide treatment can significantly reduce the infectivity of SGIV to cells, has antiviral effects against fish iridovirus, and exhibits low cytotoxicity and good safety, making it suitable for the preparation of drugs for the prevention and treatment of grouper iridovirus disease.

[0065] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of nano-zinc oxide as the sole active ingredient in the preparation of a drug against grouper iridovirus SGIV, characterized in that, The nano-zinc oxide is biosynthesized nano-zinc oxide, prepared by reducing zinc nitrate hexahydrate with Bacillus cribii; the Bacillus cribii is Bacillus cribii (… Paenibacillus kribbensis The space mutant strain △PS04-17 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 17, 2022, with accession number GDMCC No:62233.

2. The application of nano-zinc oxide as the sole active ingredient in the preparation of drugs for the prevention and treatment of grouper iridovirus disease, characterized in that, The nano-zinc oxide is biosynthesized nano-zinc oxide, prepared by reducing zinc nitrate hexahydrate with Bacillus cribii; the Bacillus cribii is Bacillus cribii (… Paenibacillus kribbensis The space mutant strain △PS04-17 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 17, 2022, with accession number GDMCC No:62233.

3. The application of nano-zinc oxide as the sole active ingredient in the preparation of drugs to alleviate or relieve grouper iridovirus disease, characterized in that, The nano-zinc oxide is biosynthesized nano-zinc oxide, prepared by reducing zinc nitrate hexahydrate with Bacillus cribii; the Bacillus cribii is Bacillus cribii (… Paenibacillus kribbensis The space mutant strain △PS04-17 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 17, 2022, with accession number GDMCC No:62233.

4. The application according to any one of claims 1 to 3, characterized in that, The drug can reduce the cytopathic effect caused by SGIV infection, decrease the expression levels of key SGIV structural protein genes MCP, VP019 and major capsid protein MCP, significantly reduce viral titer and weaken the pathogenicity of SGIV to cells.

5. The application according to any one of claims 1 to 3, characterized in that, The concentration of nano zinc oxide in the drug is 3–50 μg / mL.

6. The application according to any one of claims 1 to 3, characterized in that, The drug also contains pharmaceutically acceptable excipients.

7. The application according to any one of claims 1 to 3, characterized in that, The drug is prepared in the form of powder, capsules, or granules.

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