Use of bafilomycin a1 in the preparation of a medicament for resisting mandarin fish rhabdovirus infection

Abstract

The application discloses application of bafilomycin A1 in preparation of a medicine for resisting mandarin fish rhabdovirus infection. The application proves through experiments that bafilomycin A1 can effectively inhibit replication of SCRV, effectively inhibits virus proliferation at a cell level and a fish body level, improves the survival rate of the fish body, has important significance for prevention and treatment of the virus, and provides a useful reference for development of a new type of antiviral medicine and method.

Description

Application of Bafloxacin A1 in the Preparation of Drugs for Treating Mandarin Fish Rhabdovirus Infection Technical Field

[0001] This invention belongs to the field of aquatic organism control, specifically involving the application of bafloxacin A1 in the preparation of drugs against mandarin fish rhabdovirus infection. Background Technology

[0002] Mandarin fish (Siniperca chuatsi) is one of my country's important economic fish species, with freshwater aquaculture production exceeding 300,000 tons in recent years. Guangdong Province is the largest production area and seedling center for mandarin fish in my country, supplying over 80% of the total seedlings nationwide. With the rapid development of the mandarin fish farming industry and the gradual expansion of farming scale, various diseases affecting mandarin fish have frequently emerged, leading to severe economic losses. Among them, mandarin fish rhabdovius (Siniperca chuatsi rhabdovius, SCRV) is one of the most common and damaging diseases in mandarin fish farming. It is highly susceptible to infection from the seedling stage to the farming process, with a mortality rate exceeding 90%, especially in seedlings where the mortality rate can reach 100%. Currently, there is no effective prevention or control method.

[0003] Vacuole-ATPases (V-ATPases) are traditional proton pumps, widely found in eukaryotes from yeast to humans. In normal cells, V-ATPases are mainly expressed in the cytoplasm, where they function to maintain a neutral pH in the cytoplasm and an acidic pH in organelles. Bafilomycin A1 (BA) is a specific inhibitor of V-ATPases, inhibiting their activity. This inhibition may be related to the alteration of the extracellular acidic microenvironment caused by the suppression of V-ATPase activity. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned technical problems by providing a drug that can effectively combat mandarin fish rhabdovirus infection.

[0005] To achieve the above-mentioned objectives, this invention provides the application of bafloxacin A1 in the preparation of a drug for treating mandarin fish rhabdovirus infection.

[0006] On the other hand, the present invention also provides the use of bafloxacin A1 in the preparation of a drug for inhibiting the proliferation of mandarin fish rhabdovirus.

[0007] On the other hand, the present invention also provides the use of bafloromycin A1 in the preparation of a drug for improving the survival rate of mandarin fish.

[0008] Preferably, according to the application of the present invention, the concentration of bafloxacin A1 during application is greater than 0 and less than or equal to 5 μM.

[0009] A compound feed containing bafloxacin A1 as an active ingredient against mandarin fish rhabdovirus infection.

[0010] According to the present invention, the preparation method of the compound feed is as follows: 0.1 mg BA is dissolved in 0.5 mL DMSO, 2 mL edible oil is added to 50 g of basic feed, and then the dissolved BA is stirred evenly.

[0011] This invention demonstrates through experiments that bafloxacin A1 can effectively inhibit SCRV replication, effectively suppressing viral proliferation at both the cellular and fish body levels, and improving fish survival rates. This is of great significance for the prevention and control of the virus and provides a useful reference for the development of novel antiviral drugs and methods. Attached Figure Description

[0012] Figure 1 shows the kinetics of SCRV invasion of CPB cells.

[0013] Figure 2 shows the cell viability test results of bafloxacin A1.

[0014] Figure 3 shows the effect of bafloxacin A1 on viral replication.

[0015] Figure 4 shows the inhibitory effect of adding bafloxacin A1 at different stages of infection.

[0016] Figure 5 shows the results of a dose-dependent study on the inhibitory effect of bafloxacin A1.

[0017] Figure 6 shows the inhibitory effect of bafloxacin A1 at different MOIs of SCRV.

[0018] Figure 7 illustrates the inhibitory effect of bafloxacin A1 on SCRV infection as detected by indirect immunofluorescence.

[0019] Figure 8 shows the inhibitory effect of bafloxacin A1 on N protein expression in SCRV.

[0020] Figure 9 shows the fish's feeding behavior in response to different drugs and feed formulations.

[0021] Figure 10 shows the protective effect of bafloxacin A1 on mandarin fish after challenge.

[0022] Figure 11 shows the inhibitory effect of the combined use of amiloride and bafloxacin A1 on SCRV. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0024] 1. Materials and Methods

[0025] 1.1 Materials and Reagents

[0026] Siniperca chuatsi rhabdovirus (SCRV) and CPB cells (Chinese perch brain cell line, CPB) were isolated and preserved by the Fish Disease Laboratory of the Pearl River Fisheries Research Institute. MTT (thiazolyl blue, chemical name 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) and other reagents were purchased from Beijing TransGen Biotech Co., Ltd. Bafilomycin A1 (BA) was purchased from MCE Biotech.

[0027] 1.2 SCRV Intrusion Dynamics Experiment

[0028] To determine the dynamics of SCRV invasion of CPB cells, CPB cells were divided into groups of 3 × 10⁻⁶ cells. 5 Cells / well were seeded into 6-well plates and cultured overnight at 28°C until a monolayer was formed. SCRV was then seeded at MOI=0.01 and incubated at 4°C for 1 hour (virus binding). The cells were then washed with pre-cooled PBS and transferred to 28°C. L15 culture medium was added and cultured at 28°C. Samples were taken every hour, and the following steps were performed: (1) supernatant was collected; (2) the cells were digested with 500 μL proteinase K (1 mg / mL) for 3 min, washed three times with PBS, and 500 μL PBS was added. Cells were scraped off with a cell scraper and collected; (3) after discarding the cell supernatant, the cells were washed with PBS, and 500 μL PBS was added. Cells were scraped off with a cell scraper and collected. Nucleic acid was extracted and reverse transcribed to determine the SCRV virus copy number. Proteinase K can remove virus particles bound to the cell surface but not internalized, and detect internalized and adsorbed viruses.

[0029] N protein expression was detected by Western blotting using an anti-N protein antibody against SCRV (amino acid sequence shown in SEQ ID NO.1, synthesized by Wuhan Jinkairui Biotechnology Co., Ltd.). β-actin was used as a negative control.

[0030] Figure 1 shows the kinetics of SCRV invasion into CPB cells. A represents the kinetic curve of SCRV invasion into CPB cells, and B represents the Western spectral analysis results of N protein expression after infection. The results indicate that CPB cells were infected with SCRV (MOI=0.01), and SCRV mainly adsorbed and invaded the cells from 1 to 8 hours post-infection, reaching a peak at 8 hours (Figure 1, A). Then, 8 hours after infection, the viral load in the supernatant increased rapidly, indicating that CPB cells were destroyed and viral particles were released into the supernatant. Western spectral results showed that a large amount of virus was amplified 8 hours post-infection (Figure 1, B). These results confirm that SCRV invasion into CPB cells is a rapid process, and the optimal sampling time is 8 hours post-infection.

[0031] 1.3 Effects of MTT assay inhibitors on cell viability

[0032] The density is (3±0.3)×10 5 CPB cells were seeded at 100 μL / well in 96-well plates and incubated overnight at 28°C until a monolayer formed. 5 μM BA was then diluted with DMSO and serially diluted 2-fold, with 8 replicates per concentration. DMSO served as a negative control, and untreated cells served as a blank control. After 48 hours of incubation, 20 μL / well of MTT (0.5% w / v, dissolved in PBS) was added, and the cells were incubated for another 4 hours. The culture was then terminated, and 150 μL of DMSO was added to each well, mixed for 10 min to dissolve any crystals. The absorbance of each well was measured at OD 490 nm using an ELISA reader to calculate cell viability.

[0033] Calculate cell viability using the following formula:

[0034] Cell viability (%) = (OD) 加药 -OD 空白 ) / (OD 阴性 -OD 空白 ) × 100%.

[0035] Figure 2 shows the effect of BA on cell viability, with the control group serving as a negative control. As shown in Figure 2, the safe concentration of BA for CPB cells is below 5 μM (inclusive), and below this concentration, cell viability is not affected.

[0036] 1.4 Effect of BA on SCRV proliferation

[0037] To determine the effect of BA on viral replication, a density of (3±0.3)×10⁻⁶ was used. 5CPB cells were seeded at 100 μL / well in 12-well plates and incubated overnight at 28°C until a monolayer formed. After incubation with 5 μM BA for 1 hour, SCRV cells were seeded (MOI = 0.001). Wells without BA served as positive controls. After 24 hours of incubation, cells were observed and samples were collected. The virus solution was serially diluted 10-fold with L-15 medium, and 10 μL of each solution was used as a positive control. -1 -10 -10 Diluted viral solutions were inoculated into 96-well plates, with each concentration replicated in 8 wells. After incubation at 28°C for 1 hour, L-15 medium containing fetal bovine serum (FBS) was added to bring the final FBS concentration to 2% v / v. The plates were then incubated at 28°C for another 7 days, and the TCID was calculated using the Reed-Muench method. 50 .

[0038] Nucleic acid was extracted from the samples using the Trans series RNA extraction kit (EasyPure® RNA Kit, ER101-01, TransGold) according to the instructions, and then reverse transcribed into cDNA (EasyScript® First-Strand cDNASynthesis SuperMix, AE301-02) according to the instructions. The samples were then detected by qRT-PCR, and the SCRV virus copy number was determined using the TransGold probe fluorescence quantitative premix kit (TransScript® Probe One-Step qRT-PCR SuperMix, AQ221-01) according to the instructions.

[0039] Figure 3 shows the effect of BA on viral replication. As shown in the figure, the viral titer decreased significantly after BA treatment (left figure), and the viral copy number decreased significantly (right figure), indicating that BA can significantly inhibit SCRV replication.

[0040] 1.5 Inhibitory effects of adding BA at different stages of infection

[0041] The density is (3±0.3)×10 5 / mL of CPB cells were seeded into 12-well plates and cultured into a monolayer. The following three sets of operations were performed at each stage of infection:

[0042] (1) After adding 5 μM BA and acting for 1 hour, SCRV was inoculated (MOI=0.001).

[0043] (2) SCRV was administered at the same time as BA (MOI=0.001).

[0044] (3) Add BA 1 hour after SCRV (MOI=0.001) is inoculated.

[0045] Incubate at 28 ℃ for 24 hours, then take samples to determine the virus titer.

[0046] Figure 4 shows the inhibitory effect of adding bafloxacin A1 at different stages of infection, while the control group only received DMSO. The results showed that adding BA in advance (Pre-treatment group) and simultaneously (Co-treatment group) had better inhibitory effects, while adding BA after SCRV inoculation (Post-treatment group) had poor effects, possibly because SCRV replicates extremely rapidly and BA cannot effectively inhibit the proliferation of a large number of viruses.

[0047] 1.6 Dose-dependent study of BA inhibitory effect

[0048] The density is (3±0.3)×10 5 CPB cells were seeded at 1 / mL in 12-well plates and cultured overnight at 28°C until a monolayer formed. Different doses of BA (final concentrations of 1.25 μM, 2.5 μM, 5 μM, and 10 μM) were added and incubated for 1 hour, followed by SCRV (MOI = 0.001). Wells without BA served as positive controls. After 24 hours of incubation at 28°C, samples were collected. The titer of the samples was determined, and TCID was calculated using the Reed-Muench method. 50 .

[0049] Figure 5 shows the dose-dependent results of the bafloxacin A1 inhibitory effect. As shown in the figure, the inhibitory effect of BA on SCRV gradually increased with increasing BA dose within a certain range (1.25 μM~5 μM).

[0050] 1.7 Inhibitory effect of BA at different viral loads

[0051] The density is (3±0.3)×10 5 CPB cells were seeded at 1 / mL in 12-well plates and cultured overnight at 28°C until a monolayer formed. After incubation with BA (final concentrations of 5 μM and 2.5 μM) for 1 hour, SCRV cells were seeded (MOI = 0.001 and MOI = 0.01). Wells without BA served as positive controls. Samples were collected after 24 hours of incubation. The titer of the samples was determined, and TCID was calculated using the Reed-Muench method. 50 .

[0052] Figure 6 shows the inhibitory effect of bafloxacin A1 at different MOIs for SCRV. As shown in the figure, the inhibitory effect at MOI=0.001 is better than that at MOI=0.01.

[0053] 1.8 Indirect immunofluorescence assay to detect the inhibitory effect of BA on SCRV infection

[0054] The density is (3±0.3)×10 5CPB cells were seeded at 1 / mL in 96-well plates and cultured overnight at 28°C until a monolayer formed. After incubation with 5 μM BA for 1 hour, SCRV cells (MOI=0.001) were seeded and cultured for 8 hours. The cells were then fixed with 80% v / v acetone for 1 hour, washed three times with PBS, and incubated with PBS-diluted anti-N protein primary antibody for 1 hour. After washing three times with PBS, the cells were incubated with PBS-diluted fluorescent secondary antibody (labeled goat anti-mouse IgG (H+L); Beyotime; A0428) at room temperature for 1 hour in the dark. The cells were then washed three times with PBST buffer. For nuclear staining, PBS-diluted DAPI was added and incubated in the dark for 5 minutes, followed by washing three times with PBS to remove excess DAPI. The slides were mounted with mounting medium containing an anti-fluorescence quencher and observed under a fluorescence microscope.

[0055] Figure 7 illustrates the inhibitory effect of BA on SCRV infection as detected by indirect immunofluorescence. The results show that the viral fluorescence was significantly reduced after the addition of BA, indicating that BA has an inhibitory effect on SCRV infection.

[0056] 1.9 Inhibitory effect of BA on N protein expression in SCRV

[0057] The density is (3±0.3)×10 5 CPB cells were seeded at 1 / mL in 6-well plates and incubated overnight at 28°C until a monolayer formed. 5 μM BA was added and incubated for 1 hour, followed by SCRV (MOI=0.001). Wells seeded with SCRV without inhibitors served as positive controls, and wells without either inhibitors or SCRV seeding served as negative controls. After incubation at 28°C for 8 hours, samples were collected. N protein expression was detected by Western blotting using an N antibody against SCRV.

[0058] Figure 8 illustrates the inhibitory effect of BA on N protein expression in SCRV. The results show that BA can significantly inhibit the expression of N protein in SCRV.

[0059] 1.10 Feed Formulation

[0060] The solubility of BA in DMSO is 99 mg / mL (158.95 mM), while its solubility in water is 26.31 mg / mL (42.24 mM). Therefore, BA needs to be dissolved in DMSO to prepare the medication. However, DMSO has a pungent odor, which affects fish feeding, while adding too little DMSO will not be conducive to the uniform mixing of feed and medication.

[0061] Therefore, three groups of experiments were set up to study the optimal way to formulate feed containing BA:

[0062] (1) Group A: 0.1 mg BA was dissolved in 2 mL DMSO and then added to 50 g of feed (mandarin fish compound feed) and stirred evenly. The feeding amount was 5 g per day. The mandarin fish had a body length of 3 cm and a weight of 1.5 g.

[0063] (2) Group B: 0.1 mg BA was dissolved in 0.5 mL DMSO, and 2 mL corn oil was added to 50 g of feed. Then, the mixture was stirred evenly with the previously dissolved BA and fed to the mandarin fish.

[0064] (3) Group C: Mandarin fish were fed with ordinary feed without any added drugs as a control group.

[0065] Detecting fish feed intake: Detect the fish feed intake 1 hour after feeding, and calculate the fish feed intake by detecting the number of remaining feed particles.

[0066] Figure 9 shows the fish's feeding behavior in response to different drug and feed formulations. The results show that group B (BA+Oil) with added BA, corn oil, and trace amounts of DMSO did not affect the fish's feeding and was not significantly different from the control group (group C); while group A (BA+DMSO) used a large amount of DMSO to dissolve BA, and the excessive amount of DMSO severely affected the fish's feeding and was not conducive to drug administration.

[0067] 1.11 BA's protective effect on mandarin fish after viral challenge

[0068] Prepare the feed according to the method in Group B of "1.10 Feed Formulation" above, and feed it to the mandarin fish. Then feed the mandarin fish at a ratio of 50 LD. 50 SCRV was challenged. A negative control group (Control group) was established, consisting of mandarin fish injected with PBS, and a positive control group (SCRV group) was fed normal feed after challenge. Survival rates were calculated after 14 days of observation.

[0069] Figure 10 shows the results of the protective effect of BA on mandarin fish after challenge. As shown in the figure, the survival rate of the BA-fed group (SCRV+BA) was approximately 65%, while that of the challenge group (SCRV) was approximately 30%. This result indicates that the BA-fed group can significantly improve the survival rate of fish and effectively protect infected fish.

[0070] 1.12 Inhibitory effect of combined use of amiloride and BA on SCRV

[0071] Amiloride (EPAI, amiloride, CAS: 1428-95-1) is Na + / H +Specific inhibitors of exchange were used. Different concentrations of BA and amiloride were prepared (BA 2.5 μM + EAPI 2.5 mM, BA 1.25 μM + EAPI 2.5 mM, BA 1.25 μM + EAPI 1.25 mM, BA 1.25 μM + EAPI 0.625 mM, BA 0.625 μM + EAPI 0.625 mM) and added to cells. Cells were incubated for 1 hour, seeded with SCRV at MOI=0.001, and cultured for 24 hours. Samples were then collected. The titer of the samples was determined, and TCID was calculated using the Reed-Muench method. 50 To verify its inhibitory effect on SCRV.

[0072] The experimental results are shown in Figure 11. As can be seen from Figure 11, when BA and amiloride are used in combination, even low concentrations of EIPA and BA can synergistically enhance each other and significantly inhibit SCRV proliferation.

Claims

1. Application of bafloxacin A1 in the preparation of drugs for treating mandarin fish rhabdovirus infection.

2. Application of bafloxacin A1 in the preparation of drugs that inhibit the proliferation of mandarin fish rhabdovirus.

3. The application according to claim 1 or 2, characterized in that, Bafloxacin A1 should be applied at a concentration greater than 0 and less than or equal to 5 μM.

4. Application of bafloxacin A1 combined with amiloride in the preparation of drugs that inhibit the proliferation of mandarin fish rhabdovirus.

5. Application of bafloxacin A1 combined with amiloride in the preparation of drugs for treating mandarin fish rhabdovirus infection.

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