CN109758447B - Application of teriflunomide in preparation of drugs for preventing foot-and-mouth disease virus infection - Google Patents
Application of teriflunomide in preparation of drugs for preventing foot-and-mouth disease virus infection Download PDFInfo
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Abstract
The invention relates to an application of teriflunomide in preparation of a medicine for preventing foot-and-mouth disease virus infection, belonging to the technical field of veterinary medicines. The application of the invention can provide a high-efficiency, safe and quality-controllable anti-foot-and-mouth disease virus medicine for further controlling the spread of the foot-and-mouth disease.
Description
Technical Field
The invention relates to the technical field of veterinary medicines, and in particular relates to application of teriflunomide in preparation of a medicine for preventing foot-and-mouth disease virus infection.
Background
Foot-and-mouth disease virus is a non-enveloped single-stranded positive-strand RNA virus of the picornaviridae family. It has been found that the virus comprises 7 serotypes, I, O, C, asian I, and south africa I, ii, iii, and each serotype is divided into a plurality of subtypes. Foot-and-mouth disease caused by the virus mainly infects animals with cloven hooves such as pigs, cattle and the like, and often forms blisters in the mouth, nose, hooves and other parts, accompanied by clinical symptoms such as fever, trekking and the like. Foot-and-mouth disease has multiple transmission ways, wide epidemic range and strong infectivity, is frequently outbreak in multiple countries at present, seriously threatens the development of the global animal husbandry and has great influence on the world economy and the human society. The disease is listed as the first of a type A animal epidemic disease list by the world animal health organization, China also ranks the disease at the first of a type A animal infectious disease list, and is also one of three single diseases in the China middle and long term animal epidemic disease prevention and treatment plan (2012-2020). At present, vaccine immunization is the main means for preventing and controlling foot-and-mouth disease, however, the use of the vaccine has an "immune window period", i.e. it cannot provide protection for animals within 7 days. Therefore, in order to compensate for the "immune window period", the development of novel effective antiviral drugs is urgently needed.
Disclosure of Invention
The invention aims to provide application of Teriflunomide (Teriflunomide) in preparing a medicament for preventing foot-and-mouth disease virus infection. The application can provide a high-efficiency, safe and quality-controllable anti-foot-and-mouth disease virus medicine for further controlling the spread of the foot-and-mouth disease.
The invention provides an application of teriflunomide in preparing a medicament for preventing foot-and-mouth disease virus infection.
Preferably, the foot-and-mouth disease virus includes a type a foot-and-mouth disease virus and a type O foot-and-mouth disease virus.
Preferably, the concentration of the teriflunomide in the medicine is 200-400 mu mol/L.
The invention also provides a foot-and-mouth disease virus inhibitor which comprises teriflunomide and pharmaceutically acceptable auxiliary materials.
Preferably, the auxiliary material comprises one or more of starch, sugar powder, dextrin, polyethylene glycol and glycerol.
The invention provides an application of teriflunomide in preparing a medicament for preventing foot-and-mouth disease virus infection. The medicament prepared by taking teriflunomide as an effective component can inhibit foot-and-mouth disease virus (FMDV) and prevent and control the foot-and-mouth disease. Cell tests show that teriflunomide has low cytotoxicity and has an inhibiting effect on replication of A-type foot-and-mouth disease viruses and O-type foot-and-mouth disease viruses; further experiments confirmed that teriflunomide only works early in FMDV replication and does not prevent viral replication when it enters late stage of viral replication. Teriflunomide can be used as an effective anti-foot-and-mouth disease virus component.
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FIG. 1 is a graph of the cytotoxicity of different concentrations of Teriflunomide against IBRS-2 cells in example 1 of the present invention;
FIG. 2 is a graph showing the inhibitory effect of Teriflunomide at various concentrations in example 1 of the present invention on infection of type O FMDV with IBDS-2 cells;
FIG. 3 is a graph showing the inhibitory effect of different concentrations of Teriflunomide in example 1 of the present invention on type A FMDV infection of IBDS-2 cells;
FIG. 4 is a graph showing the effect of Teriflunomide at various concentrations on the inhibition of FMDV mRNA in type-O FMDV-infected cells in example 1 of the present invention;
FIG. 5 is a graph showing the effect of Teriflunomide at different concentrations on the inhibition of VP1 protein expression in type O FMDV-infected cells in example 1 of the present invention;
FIG. 6 is a graph showing the effect of IFA detection of different concentrations of Teriflunomide in the inhibition of FMDV protein expression in type-O FMDV-infected cells in example 1 of the present invention;
FIG. 7 is a graph showing the effect of Teriflunomide on the inhibition of FMDV mRNA in virus-infected cells at various time periods in example 1 of the present invention;
FIG. 8 is a graph showing the inhibitory effect of Teriflunomide on the expression of VP1 protein in virus-infected cells at different time periods in example 1 of the present invention;
FIG. 9 is a graph showing the effect of Teriflunomide on the death time of FMDV-infected suckling mice in example 1 of the present invention.
Detailed Description
The invention provides an application of teriflunomide in preparing a medicament for preventing foot-and-mouth disease virus infection. In the present invention, the foot-and-mouth disease virus includes a type a foot-and-mouth disease virus and a type O foot-and-mouth disease virus. The teriflunomide has an inhibiting effect on cytopathic effect induced by both foot and mouth disease virus A and foot and mouth disease virus O, and inhibits the replication of the viruses, wherein the teriflunomide only acts in the early replication stage of FMDV, and cannot prevent the replication of the viruses when entering the later replication stage of the viruses. The medicine prepared by taking teriflunomide as an effective component has low cytotoxicity, has an inhibition effect on the replication of A-type foot-and-mouth disease virus and O-type foot-and-mouth disease virus, and can inhibit foot-and-mouth disease virus (FMDV) and prevent and control foot-and-mouth disease. The source of teriflunomide in the invention is not particularly limited, and conventional commercial products in the field can be adopted.
In the invention, the concentration of the teriflunomide in the medicine is preferably 200-400 mu mol/L, and more preferably 400 mu mol/L.
The invention also provides a foot-and-mouth disease virus inhibitor which comprises teriflunomide and pharmaceutically acceptable auxiliary materials.
In the invention, the auxiliary material comprises one or more of starch, powdered sugar, dextrin, polyethylene glycol and glycerol. In the present invention, the glycerin is preferably glycerin for injection. The sources of the auxiliary materials in the present invention are not particularly limited, and commercially available products of conventional starch, sugar powder, dextrin, polyethylene glycol and glycerin, which are well known to those skilled in the art, may be used.
The application of teriflunomide in the preparation of a medicament for preventing foot and mouth disease virus infection is further described in detail with reference to specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
1. Experimental Material
1.1 cells, animals, viruses and drugs
IBRS-2 cells were preserved from this group; the 3-day-old suckling mice were purchased from the laboratory animal farm of the Lanzhou veterinary research institute of the Chinese academy of agricultural sciences. FMDV (O/MY98/BY/2010 and A/GDMM/CHA/2013) is preserved and provided BY the national foot-and-mouth disease reference laboratory; teriflunomide was purchased from MCE and formulated in DMSO.
1.2 reagents
DMEM, fetal bovine serum FBS, trypsin medium were purchased from Gibco; MTS assay kit was purchased from Abcam corporation; TRIZOL was purchased from Invitrogen; SYBRPremix Ex TaqⅡThe kits are purchased from precious bioengineering (Dalian) Co., Ltd; RIPA lysate, BCA method protein quantification kit, SDS-PAGE gel preparation kit and ECL from Biyuntian company; BSA, PVDF membranes were purchased from BioRad; tween-20 and Tween-80 are purchased from Shanghai Bioengineering company; triton X-100, DMSO was purchased from Sigma; mouse anti-beta-actin polyclonal antibody, HRP marked anti-rabbit or anti-mouse IgG antibody are purchased from Abcam company; the rabbit anti-O type FMDVVP1 polyclonal antibody is a gift from Zhenghai doctor in national foot and mouth disease reference laboratory; high resistance of rabbit to type O FMDVThe immune serum is a gift in the national foot and mouth disease reference laboratory.
2. Experimental methods and results
2.1 toxicity assay of Teriflunomide on IBRS-2 cells:
the cytotoxicity of Teriflunomide on IBRS-2 cells was determined by MTS method. After the cells are paved on a 96-pore plate IBRS-2 and fully grow into a monolayer, the culture solution on the upper layer of the cells is discarded, the cells are washed for 3 times by using fresh DMEM, finally 100 mu L of the Teriflunomide diluted by the DMEM culture solution containing 2% FBS in a gradient manner is added, the corresponding DMSO concentration of the preparation solution of the Teriflunomide is used as a negative control hole, and the cells are used as a cell control hole without any treatment. The cells were incubated at 37 ℃ for 72h, the supernatant cell culture was discarded, washed three times with fresh DMEM, and 100. mu.L of fresh DMEM was added, and 20. mu.L of LMTS solution was added to each well. After incubation at 37 ℃ for 4h, the absorbance at 490nm was measured on a microplate reader, according to the formula "cell activity rate ═ ODMedicine-ODBlank space)/(ODNegative of-ODBlank space) X 100% "toxicity of teriflumide at different concentrations to IBRS-2 cells was calculated. The experiment was independently repeated three times.
The experimental results are shown in fig. 1: MTS results show that with increasing drug concentration, the toxicity to IBRS-2 cells increases. When the concentration is 400. mu. mol or less, the cell activity is 70% or more. When the concentration of the drug is higher than 400. mu. mol, the activity of the cells decreases rapidly, and at a concentration of 1000. mu. mol, the activity rate of the cells is 20%. Therefore, when the anti-FMDV is prepared using this drug, its concentration should not be higher than 400. mu. mol.
2.2 evaluation of Teriflunomide against foot-and-mouth disease Virus Activity on IBRS-2 cells:
well-grown IBRS-2 cells on a DMEM complete medium containing 10% FBS are paved on a 96-well plate, after the IBRS-2 cells grow to a full monolayer, cell upper layer culture solution is discarded, the cells are washed 3 times BY fresh DMEM, and 100TCID50O/MY98/BY/2010 is inoculated. After 1h, the virus solution was removed, washed 3 times with fresh DMEM, 100. mu.L of Teriflunomide diluted in gradient with 2% FBS-containing DMEM medium was added, the DMSO concentration corresponding to the preparation solution of Teriflunomide was used as virus control well, and Teriflunomide-free, virus-free samples were used as virus control wellsUsed as a cell control well. The cells were incubated at 37 ℃ for 24h, the supernatant was discarded, washed three times with fresh DMEM, 100. mu.L of fresh DMEM was added, and 20. mu.L of LMTS solution was added to each well. After incubation at 37 ℃ for 4h, the absorbance at 490nm was measured on a microplate reader, according to the formula "cell activity rate ═ ODMedicine-ODBlank space)/(ODNegative of-ODBlank space) X 100% "the antiviral effect of teriflumide at different concentrations was calculated. Different groups of supernatants were collected simultaneously and q-PCR and Western Blot were used to detect the mRNA and FMDVVP1 protein levels of FMDV2B gene, respectively. RNA of the cells was extracted according to TRIZOL instructions, and fluorescent quantitative PCR was performed according to SYBR Premix Ex Taq II instructions, and β -actin was used as an internal reference gene. The primer sequence for detecting the mRNA specificity of the FMDV2B gene is as follows:
FMDV-for,5’-CAACAAAACACGGACCCGAC-3’(SEQ ID NO.1)；
FMDV-rev,5’-TTGTACCAGGGTTTGGCCTC-3’(SEQ ID NO.2)；
the primer sequence of beta-actin is as follows:
β-actin for,5’-GACCACCTTCAACTCGATCA-3’(SEQ ID NO.3)；
beta-actin-rev, 5'-GTGTTGGCGTAGAGGTCCTT-3' (SEQ ID NO. 4). The reaction system is as follows: SYBR Premix ExTaq: 12.5. mu.L, upstream primer: 1 μ L, downstream primer: 1 μ L, cDNA: 1 μ L, sterilized water: 9.5. mu.L, the reaction program is: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 56 for 30s, and extension at 72 ℃ for 30s, for 40 cycles. According to
The method calculates the expression level of the sample relative to the reference gene. Extracting protein with protein lysate, and determining the concentration of the extracted protein by using BCA method. Preparing 12% separation gel to carry out protein SDS-PAGE denaturing electrophoresis, and after 2 hours of electrophoresis, electrically transferring the protein to a PVDF membrane. After the membrane is transferred for 2 hours, the membrane is put into 5 percent of freshly prepared skim milk powder for sealing for 1 hour. After blocking, the membrane was placed in rabbit anti-O FMDV VP1 polyclonal antibody (1:3000) and mouse anti-beta-actin polyclonal antibody (1:4000) and incubated overnight in a refrigerator at 4 ℃. Washing the membrane with TBST for 10min 5 times, and placing the membrane into corresponding secondary antibody HRP-labeled goatAnti-rabbit IgG, goat anti-mouse IgG labeled with HRP (1:3000), incubating at room temperature for 1h, washing the membrane for 5 times with TBST, each time for 10min, and finally detecting FMDV VP1 protein by ECL chemiluminescence method. In order to study whether Teriflunomide has an inhibiting effect on other foot-and-mouth disease virus subtypes, 100TCID50A/GDMM/CHA/2013 is used for infecting cells, and the MTS method is used for determining the antiviral activity of the Teriflunomide.
The experimental results are shown in FIGS. 2 to 5: the results of testing whether Teriflunomide has antiviral activity against FMDV with MTS showed that Teriflunomide can provide more than 70% protection for IBDV-2 cells (FIG. 2) and significantly inhibit the expression of FMDV mRNA (FIG. 4) and VP1 levels (FIG. 5) at a concentration of 400. mu. mol when different concentrations of the drugs are added. While at concentrations below 400. mu. mol, Teriflunomide failed to provide effective cell protection and reduced expression of FMDV mRNA and VP1 levels. Similarly, 200, 400. mu. mol of Teriflunomide was effective in protecting IBDS-2 cells when infected with type A foot-and-mouth disease virus (FIG. 3), indicating that Teriflunomide also has antiviral activity against type A FMDV.
2.3 Indirect immunofluorescence assay for FMDV protein expression in infected cell groups
The density is 3 x 105And (3) paving the/-hole IBRS-2 cells on a 12-hole plate, discarding culture solution on the upper layer of the cells after the IBRS-2 cells grow to be full of a monolayer, washing the cells with fresh DMEM for 3 times, and inoculating 100TCID50O/MY 98/BY/2010. After 1h, the virus solution was removed, washed 3 times with fresh DMEM, 100. mu.L of Teriflunomide diluted in gradient with 2% FBS-containing DMEM was added, and the virus control wells, which had the DMSO concentration corresponding to the preparation solution of Teriflunomide, were placed in the cells and incubated at 37 ℃ for 12 h. Discarding the upper cell culture solution, washing with PBS for 2 times, fixing cells with 4% paraformaldehyde for 15min, discarding paraformaldehyde, adding methanol for 5min, rinsing with PBS for 3 times, 5min each time, adding blocking solution (10% FBS, 0.3% Triton X-100, 89.7% PBS) for blocking for 10min, adding primary antibody (1:100) diluted with blocking solution, incubating at room temperature for 1h, rinsing with PBS for 3 times, 5min each time, adding secondary antibody (1:200) diluted with blocking solution, incubating at room temperature for 1h, rinsing with PBS for 5 times, 5min each time. Finally adding 300 mu LDAPI into each hole for staining, acting for 5min, rinsing with PBS for 2 times, each time for 5min, observing with fluorescence microscopeAnd (6) obtaining the result.
The experimental results are shown in fig. 6: a large amount of specific fluorescence was observed in the untreated virus-infected IBRS-2 cells and virus-infected groups treated with 50, 100, 200. mu. mol of Teriflunomide, whereas a small amount of fluorescence was observed in the 400. mu. mol of Teriflunomide-treated IBRS-2 cells. This result further demonstrates that Teriflunomide exhibits dose-dependent anti-foot-and-mouth disease virus activity on IBRS-2 cells.
2.4 evaluation of inhibition time of foot-and-mouth disease virus infected IBRS-2 cells by Teriflunomide:
well-grown IBRS-2 cells on DMEM complete medium containing 10% FBS were plated on a 12-well plate, after the IBRS-2 cells grew to a full monolayer, the cell supernatant was discarded, washed 3 times with fresh DMEM, and inoculated with 100TCID50O/MY 98/BY/2010. After 1h, the virus solution was removed, washed 3 times with fresh DMEM, and DMEM with 2% FBS was added as 0 h. Teriflunomide was added to different wells at 0h, 2h, 4h, 8h and 16h after virus infection to give a final concentration of 400. mu. mol. And meanwhile, a negative control without adding the medicine is set. Culturing in a constant-temperature cell culture box with CO2 at 37 ℃ for 48 h. Different groups of supernatants were collected and q-PCR and Western Blot were used to detect FMDV2B gene mRNA and FMDVVP1 protein levels, respectively.
The experimental results are shown in FIGS. 7 to 8: cells were treated with teiunfunomide at different time periods after viral infection and the results showed significant inhibition of FMDV mRNA levels (figure 7) and VP1 protein levels (figure 8) compared to the negative control within 0-8 h of FMDV replication. The inhibition effect of Teriflunomide at 16h is not obvious, which indicates that Teriflunomide only acts early in FMDV replication and cannot prevent virus replication when entering the later stage of virus replication.
2.5 evaluation of the anti-foot-and-mouth disease virus activity of Teriflunomide in vivo:
24 SPF BALB/C suckling mice of 3 days old are randomly divided into two groups, 12 mice/group, and the suckling mice and the mother mice are raised together. The experiment is divided into two groups, wherein the neck of 12 suckling mice in the experimental group is injected with 60 mu g of Teriflunomide subcutaneously, and the whole neck of 12 suckling mice in the control group is inoculated with 100 mu L of PBS containing 10% Tween-80 subcutaneously. After 2h, all pups were injected subcutaneously in the neck with 100. mu.L PBS containing 100LD50O/MY 98/BY/2010. The death of the suckling mice was continuously observed and recorded. In order to ensure the safety of the experiment, the toxicity of the animals is attacked in a P3 laboratory, and the related operations conform to the related regulations of the Experimental animal management Committee of the Chinese academy of agricultural sciences.
The results of the experiment are shown in FIG. 9: the control group of suckling mice died from 30h after virus infection, and all died at 54h, while the experimental group died at 60h and all died at 102h, and the death time was delayed by 48h compared with the control group. Therefore, the Teriflunomide can obviously delay the lethal effect of the foot-and-mouth disease virus on suckling mice.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
Application of teriflunomide in preparation of drugs for preventing foot-and-mouth disease virus infection
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<170> SIPOSequenceListing 1.0
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Claims (2)
1. The application of teriflunomide in preparing a medicament for preventing foot-and-mouth disease virus infection is characterized in that the foot-and-mouth disease virus is A type foot-and-mouth disease virus and O type foot-and-mouth disease virus.
2. The use according to claim 1, wherein the concentration of teriflunomide in the medicament is 200-400 μmol/L.
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| WO2017117006A1 (en) * | 2015-12-29 | 2017-07-06 | The Board Of Trustees Of The Leland Stanford Junior University | Use of a dhodh inhibitor in combination with an inhibitor of pyrimidine salvage |
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| CN108721281A (en) * | 2017-04-20 | 2018-11-02 | 华东理工大学 | New antiviral drugs and its application |
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| CN106880608A (en) * | 2015-12-15 | 2017-06-23 | 北大方正集团有限公司 | A kind of teriflunomide dispersible tablet and preparation method thereof |
| WO2017117006A1 (en) * | 2015-12-29 | 2017-07-06 | The Board Of Trustees Of The Leland Stanford Junior University | Use of a dhodh inhibitor in combination with an inhibitor of pyrimidine salvage |
| CN108721281A (en) * | 2017-04-20 | 2018-11-02 | 华东理工大学 | New antiviral drugs and its application |
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