Application of harringtonine in preparation of anti-Seneca virus preparation

Abstract

The invention provides application of harringtonine in preparation of anti-Seneca virus preparations, and belongs to the technical field of preparation of antiviral drugs. The invention provides new application of harringtonine, and harringtonine can effectively inhibit replication of Seneca virus and reduce activity of Seneca virus, and can be used for preparing anti-Seneca virus preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of antiviral drug preparation, and particularly relates to an application of harringtonine in the preparation of an anti-Seneca virus preparation. Background Art

[0002] Seneca Valley Virus (SVV) is a member of the picornavirus family and exhibits typical picornavirus characteristics. The virus particles are spherical, icosahedral, and lack an envelope. The SVV genome is a single-stranded, positive-sense RNA (SNS) approximately 7.2 kb in length, consisting of two noncoding regions (5′ UTR and 3′ UTR) and an open reading frame (ORF). Pigs are the sole natural host of SVV infection. Infection causes Seneca virus disease, characterized by vesicular lesions on the snout, hoof crown, and hoof fork. Its clinical symptoms are highly similar to those of other swine vesicular diseases (such as foot-and-mouth disease and swine vesicular disease), making them difficult to distinguish. SVV is also associated with sudden mortality in newborn piglets. The disease is spreading globally, particularly in the United States and Brazil, where swine production is concentrated, resulting in multiple outbreaks and significant economic losses to the pig industry. Currently, there is no safe and effective vaccine or treatment for Seneca virus disease. Therefore, the development of safe and effective antiviral drugs is crucial for epidemic prevention and control.

[0003] Harringtonine (HT) is a natural alkaloid first isolated from Cephalotaxus in 1963. HT has multiple biological functions, including inhibiting protein synthesis by blocking peptide bond formation and aminoacyl-tRNA binding; activating the caspase pathway and disrupting mitochondrial membrane potential, promoting apoptosis; and inducing cell arrest in the G1 phase, inhibiting cell proliferation. It also plays a significant role in anti-tumor activities and is used to treat acute myeloid leukemia and chronic myeloid leukemia. The present invention has newly discovered anti-Seneca virus activity and can be used to prepare anti-Seneca virus preparations. Summary of the Invention

[0004] The purpose of the present invention is to provide a new use of harringtonine for preparing an anti-Seneca virus disease preparation.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A drug for Seneca virus disease containing harringtonine.

[0006] Application of harringtonine in preparing anti-Seneca virus preparations.

[0007] The use as described above is characterized in that the effective concentration of the harringtonine in the anti-Seneca virus preparation is 1-30 μM.

[0008] The use as described above is characterized in that the effective concentration of harringtonine in the anti-Seneca virus preparation is 1 μM, 10 μM, or 20 μM.

[0009] The beneficial effects of the present invention are: The present invention provides a new use of harringtonine for in vitro anti-Seneca virus. Harringtonine can effectively inhibit the replication of Seneca virus and reduce its activity, and can be used to prepare anti-Seneca virus preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The effect of different concentrations of harringtonine on the viability of BHK-21 cells; Figure 2 The effect of different concentrations of harringtonine on the viability of PK-15 cells; Figure 3 The effect of different concentrations of harringtonine on the proliferation of recombinant SVV strain rSVV-eGFP; Figure 4 Western blot was used to detect the effects of different concentrations of harringtonine on SVV replication (expression of viral capsid protein VP3) (BHK-21 cells); Figure 5 Western blot was used to detect the effects of different concentrations of harringtonine on SVV replication (expression of viral capsid protein VP3) (PK-15 cells); Figure 6 The effects of different concentrations of harringtonine on SVV titer were detected by TCID50 (BHK-21 cells); Figure 7 The effects of different concentrations of harringtonine on SVV titer were detected by TCID50 (PK-15 cells); Figure 8 Western blot was used to detect the effect of harringtonine on the expression of viral capsid protein VP3 at different time points after SVV infection (BHK-21 cells); Figure 9 Western blot was used to detect the effect of harringtonine on the expression of viral capsid protein VP3 at different time points after SVV infection (PK-15 cells); Figure 10 The effect of harringtonine on the proliferation curve of SVV was detected by TCID50 (BHK-21 cells); Figure 11 The effect of harringtonine on the proliferation curve of SVV was detected by TCID50 (PK-15 cells). DETAILED DESCRIPTION

[0011] The present invention explores the regulatory effect of harringtonine on SVV replication in vitro and finds that harringtonine can be used to prepare drugs against Seneca virus.

[0012] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all fall within the scope of the present invention.

[0013] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the examples are of analytical grade or above, and commercially available products can be used, such as DMEM culture medium and fetal bovine serum can be purchased from Thermo Fisher Scientific; trypsin can be purchased from Amresco Co., Ltd., goat anti-mouse IgG (H+L) HRP can be purchased from Solebro Co., Ltd., goat anti-rabbit IgG (H+L) HRP and nitrocellulose membrane (NC membrane) can be purchased from Bio-Rad Co., Ltd.; FITC-labeled goat anti-mouse IgG (H+L) can be purchased from Merck Co., Ltd., protein Marker can be purchased from Yazyme Co., Ltd., SDS-PAGE Buffer (5×) can be purchased from Kangwei Century Co., Ltd., methanol can be purchased from Fuyu Fine Chemical Co., Ltd., skimmed milk powder, tris (hydroxymethyl)aminomethane (Tris Base), peptone, yeast powder, agarose, sodium dodecyl sulfate (SDS), 50× TAE buffer, green fluorescent nucleic acid dye, 5× loading buffer, and transfection reagent lip2000 can be purchased from MCE Co., Ltd., RNA extraction kit can be purchased from Yisheng Biotechnology Co., Ltd., reverse transcription kit can be purchased from Langbolid Biotechnology Co., Ltd., and SDS-PAGE gel preparation kit can be purchased from Yazyme Co., Ltd.

[0014] Example 1 1. Materials The BHK-21 (hamster kidney cells), PK-15 (pig kidney cells), and SVV CHhb17 strain used in the present invention were provided by the laboratory of the High-Tech Research Laboratory of Livestock and Poultry Biological Preparations, Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences. The SVV recombinant virus rSVV-eGFP expressing green fluorescent protein was donated by Professor Liu Fuxiao of Qingdao Agricultural University.

[0015] The main chemical reagents and preparation methods used in the solution are shown in Table 1.

[0016] Table 1 Main chemical reagents and preparation methods 2 Methods and Results 2.1 Cell culture In this study, BHK-21 and PK-15 cells were mainly used as host cells for SVV infection.

[0017] 2.1.1 Cell recovery Prepare a 37°C water bath in advance and quickly thaw the cells. Thaw the cells in approximately 1 minute, until the cryopreservation solution in the tube is completely thawed. Centrifuge the cells at a low speed of 1000 rpm for 3 minutes. Carefully aspirate the supernatant with a pipette, add culture medium to 5 mL, and mix thoroughly by pipetting. Gently shake the cell culture flask to ensure even distribution of cells, then place the flask in an incubator for culture.

[0018] 2.1.2 Cell passaging When the cell density reaches 90% or above, cells can be passaged. Discard the old culture medium and gently add phosphate-buffered saline (PBS) to the cell wells to wash the cells. Then, digest with 0.25% trypsin. When the cells begin to detach by naked eye observation, pour off the supernatant and vigorously tap the side of the cell flask to make the cells fall like quicksand. Then, add an appropriate amount of culture medium, pipette the cells evenly, and transfer them to a cell well plate for subsequent experiments.

[0019] 2.1.3 Cell cryopreservation The cells were collected by centrifugation into a centrifuge tube, and freshly prepared cell freezing solution was added. The cells were first placed in a -80°C refrigerator for 24 h, and then transferred to a liquid nitrogen tank for long-term storage.

[0020] 2.2 Determination of cytotoxicity of harringtonine To determine the cytotoxic effects of harringtonine, cells were treated with harringtonine at final concentrations of 0 μM (mock), 1 μM, 10 μM, and 20 μM, and cell viability was assessed using the CCK-8 assay. Specifically, cells were seeded at an appropriate density in a 96-well culture plate. The plate was gently shaken to evenly distribute the cells and then placed in a cell incubator for incubation. After 24 hours, 1 μM, 10 μM, and 20 μM harringtonine were added. After 24 hours, the CCK-8 reagent was added, taking care to avoid air bubbles. After incubation in the incubator for 1 hour, the absorbance at 450 nm was measured to calculate cell viability.

[0021] The viability results of BHK-21 cells were as follows Figure 1 As shown, the viability results of PK-15 cells are as follows Figure 2 As shown in the figure, it can be seen that the activities of BHK-21 and PK-15 cells treated with these three concentrations of harringtonine are no different from those of the control group, indicating that harringtonine at concentrations of 20 μM and below has no significant effect on the activities of both cell lines.

[0022] 2.3 Effect of harringtonine on SVV replication 2.3.1 Effects of different doses of harringtonine on SVV replication To evaluate the effects of different doses of harringtonine on SVV replication, this study first used fluorescence microscopy to observe the effects of 0 μM, 1 μM, 10 μM, and 20 μM treatment on eGFP-labeled recombinant SVV. Specifically, BHK-21 cells were plated in 6-well plates and cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. When the cells reached 70-80% confluency, they were inoculated with a recombinant SVV expressing green fluorescent protein, rSVV-eGFP, at a multiplicity of infection (MOI) of 5. Harringtonine was then added to the cells to give final concentrations of 0 μM, 1 μM, 10 μM, and 20 μM. Samples were collected 12 hours after infection, and fluorescence intensity was analyzed using fluorescence microscopy.

[0023] The results are as follows Figure 3 As shown, the results showed that with the increase of the dose of harringtonine, the rSVV-eGFP fluorescence signal gradually decreased, indicating that the viral replication was inhibited.

[0024] To further determine the effects of different doses of harringtonine on SVV replication, Western blot and TCID50 assays were used to analyze the effects of different doses of harringtonine on wild-type SVV CHhb17 capsid protein expression and viral titer. Specifically, BHK-21 and PK-15 cells were plated in 6-well plates and cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2. When the cells reached 70-80% confluency, SVV was inoculated at an MOI of 1, and harringtonine was added at final concentrations of 0 μM, 1 μM, 10 μM, and 20 μM. Cells were harvested 12 hours after infection, and viral capsid protein expression and viral titer were analyzed by Western blot and TCID50 assays, respectively.

[0025] Western blot results of BHK-21 cells are as follows Figure 4 As shown, the Western blot results of PK-15 cells are as follows Figure 5 As shown, the TCID50 results of BHK-21 cells are as follows Figure 6 The TCID50 results of PK-15 cells are shown in Figure 7 As shown, from Figure 4-7 As can be seen, 1 μM, 10 μM, and 20 μM harringtonine treatment of cells can inhibit the expression of viral capsid protein VP3 12 h after SVV infection ( Figure 4 and Figure 5 ) and virus titer ( Figure 6 and Figure 7 ).

[0026] 2.3.2 Effects of harringtonine on SVV replication at different stages of infection Based on the above experiments, we further analyzed the effects of harringtonine on SVV replication at different stages of infection. Cells were harvested at different time points after infection, and the effects of harringtonine on wild-type SVV CHhb17 capsid protein expression and viral titer were analyzed by Western blot and TCID50 assay. Specifically, BHK-21 and PK-15 cells were plated in six-well plates and cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2. When cells reached 70-80% confluency, SVV CHhb17 was inoculated at an MOI of 1, and harringtonine was added to a final concentration of 20 μM. Cells were harvested at 0, 6, 9, and 12 hours post-infection, and viral capsid protein VP3 expression (β-actin was used as an internal reference protein) and viral titer were analyzed by Western blot and TCID50 assay, respectively.

[0027] Western blot results are as follows Figure 8 and Figure 9 As shown in the results, the harringtonine-treated group could inhibit the expression of viral protein VP3 at 9 h and 12 h after virus infection compared with its solvent control DMSO-treated group); the virus titer test results are shown in Figure 10 and Figure 11 As shown in the figure, *** indicates significant difference (P < 0.01). The results showed that harringtonine treatment significantly inhibited SVV replication 6 h, 9 h and 12 h after SVV infection, with the virus titer decreasing 10-fold at 6 h, 8.91-fold at 9 h, and 3.76-fold at 12 h.

[0028] In summary, harringtonine can significantly inhibit SVV replication and has anti-SVV activity. Harringtonine can be used to prepare anti-SVV drugs.

Claims

1. An anti-Seneca virus disease drug, characterized in that: It contains harringtonine.

2. Application of harringtonine in the preparation of anti-Seneca virus preparations.

3. The use according to claim 2, characterized in that The effective concentration of the harringtonine in the anti-Seneca virus preparation is 1-30 μM.

4. The use according to claim 2, characterized in that The effective concentration of harringtonine in the anti-Seneca virus preparation is 1 μM, 10 μM, and 20 μM.

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