CN109793741B - Application of halofuginone in preparation of drugs for preventing foot-and-mouth disease virus infection - Google Patents
Application of halofuginone in preparation of drugs for preventing foot-and-mouth disease virus infection Download PDFInfo
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- CN109793741B CN109793741B CN201910202391.8A CN201910202391A CN109793741B CN 109793741 B CN109793741 B CN 109793741B CN 201910202391 A CN201910202391 A CN 201910202391A CN 109793741 B CN109793741 B CN 109793741B
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Abstract
The invention relates to an application of halofuginone in preparing a medicament for preventing foot-and-mouth disease virus infection, belonging to the technical field of veterinary medicaments. The application of the invention can provide a high-efficiency, safe and quality-controllable medicament for resisting foot-and-mouth disease virus 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 halofuginone 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. 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 Halofuginone in preparation of 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 application of halofuginone in preparation of a medicine 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 said halofuginone in said medicament is > 1 μmol/L.
The invention provides application of halofuginone in preparation of a medicine for preventing foot-and-mouth disease virus infection. The halofuginone has inhibitory effect on cytopathic effect induced by type A foot-and-mouth disease virus and type O foot-and-mouth disease virus, and can inhibit virus replication. Cell tests show that halofuginone has low cytotoxicity and has an inhibiting effect on replication of A-type foot-and-mouth disease virus and O-type foot-and-mouth disease virus; further experiments confirmed that halofuginone only works early in FMDV replication and does not prevent viral replication when it enters late stage of viral replication. Halofuginone 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 Halofuginone at various concentrations on IBRS-2 cells in example 1 of the present invention;
FIG. 2 is a graph showing the inhibitory effect of Halofuginone at various concentrations on FMDV type O infection of IBDS-2 cells in example 1 of the present invention;
FIG. 3 is a graph showing the inhibitory effect of Halofuginone at various concentrations on FMDV type A infected IBDS-2 cells in example 1 of the present invention;
FIG. 4 is a graph showing the effect of Halofuginone at various concentrations on FMDV mRNA inhibition in type O FMDV infected cells in example 1 of the present invention;
FIG. 5 is a graph showing the effect of Halofuginone in different concentrations on the inhibition of VP1 protein expression in cells infected with type O FMDV in example 1 of the present invention;
FIG. 6 is a graph showing the effect of IFA detection of Halofuginone at different concentrations on 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 q-PCR detection of Halofuginone on the inhibition of mRNA during the FMDV adsorption and penetration stages in example 1 of the present invention;
FIG. 8 is a graph of the inhibitory effect of Halofuginone on FMDVmRNA of virally infected cells at various time periods in example 1 of the present invention;
FIG. 9 is a graph showing the inhibitory effect of Halofuginone on VP1 protein expression in virus-infected cells at different time periods in example 1 of the present invention.
Detailed Description
The invention provides application of halofuginone in preparation of a medicine 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 halofuginone has an inhibiting effect on cytopathic effect induced by both A-type foot-and-mouth disease virus and O-type foot-and-mouth disease virus, and can inhibit the replication of the virus, wherein the halofuginone only plays a role in the early replication stage of FMDV, but cannot prevent the replication of the virus when entering the later replication stage of the virus. The source of halofuginone in the present invention is not particularly limited, and any conventional commercially available product in the art may be used.
In the present invention, the concentration of said halofuginone in said drug is > 1. mu. mol/L.
The application of halofuginone 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, viruses and drugs
IBRS-2 cells were preserved from this group; FMDV (O/MY98/BY/2010 and A/GDMM/CHA/2013) is preserved and provided BY the national foot-and-mouth disease reference laboratory; halofuginone 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; SYBR Premix Ex Taq II kits were purchased from Bao 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 was purchased from shanghai bio-engineering; 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 FMDV VP1 polyclonal antibody is a gift offered by Zhenghai doctor in national foot and mouth disease reference laboratory; the rabbit anti-O type FMDV hyper-immune serum is a gift in China foot and mouth disease reference laboratory.
2. Experimental methods and results
2.1 toxicity assay of Halofuginone on IBRS-2 cells:
the cytotoxicity of Halofuginone on IBRS-2 cells is determined by MTS method. After the cells are paved on a 96-pore plate IBRS-2 and fully grow into a monolayer, the upper layer culture solution of the cells is discarded, the cells are washed for 3 times by using fresh DMEM, finally 100 mu L of Halofuginone which is diluted by the DMEM culture solution containing 2% FBS in a gradient mode is added, the corresponding DMSO concentration of the preparation solution of the Halofuginone 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 72 hours, 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 MTS 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 Halofuginone at different concentrations on IBRS-2 cells was calculated. The experiment was independently repeated three times.
The experimental results are shown in fig. 1: MTS results show that the activity rate of cells is still above 80% with the continuous increase of the concentration of the drug, which indicates that Halofuginone has extremely low cytotoxicity to IBRS-2.
2.2 evaluation of Halofuginone activity against foot and mouth disease Virus 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 100TCID 50O/MY 98/BY/2010 is inoculated. After 1h, the virus solution is removed, the cell is washed 3 times by fresh DMEM, 100 mu L of Halofuginone which is diluted by DMEM culture solution containing 2% FBS in a gradient manner is added, the corresponding DMSO concentration of the preparation solution of Halofuginone is used as a virus control hole, and the cell without Halofuginone and virus is used as a cell control hole. The cells were incubated at 37 ℃ for 48h, 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 MTS solution was added to each well. At 490nm measured on a microplate reader after incubation for 4h at 37 ℃Absorbance value according to the formula "cell activity rate ═ ODMedicine-ODBlank space)/(ODNegative of-ODBlank space) X 100% "the antiviral action of Halofuginone at different concentrations was calculated. At the same time, different groups of supernatants were collected, and q-PCR and WesternBlot tested for FMDV 2B gene mRNA and FMDV VP1 protein levels, 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 specificity of FMDV 2B gene mRNA 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, then placing the membrane into corresponding secondary antibody HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG (1:3000), incubating at room temperature for 1h, washing the membrane with TBST for 10min 5 times, and finally, developing and detecting FMDV VP1 protein by ECL chemiluminescence method. To investigate whether Halofuginone has activity against type A foot-and-mouth disease virusInhibition, 100TCID50A/GDMM/CHA/2013 infected cells, MTS method for determining antiviral activity.
The experimental results are shown in FIGS. 2 to 5: the results of using MTS to test whether Halofuginone has antiviral activity against FMDV show that Halofuginone can provide more than 80% protection of IBRS-2 cells at concentrations greater than 0.5. mu. mol (FIG. 2) and significantly inhibit the expression of FMDV mRNA and VP1 protein levels (FIG. 4, FIG. 5) when different concentrations of drugs are added respectively. Whereas Halofuginone does not provide effective protection of cells at concentrations of 0.5 μmol. Similarly, when cells were infected with type A foot and mouth disease virus, 1. mu. mol and above of Halofuginone was effective in protecting IBRS-2 cells (FIG. 3), indicating that Halofuginone 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, removing the culture solution on the upper layer of the cells after the IBRS-2 cells grow to be full of a monolayer, washing the cells for 3 times BY using fresh DMEM, and inoculating 100TCID 50O/MY 98/BY/2010. After 1h, the virus solution was removed, washed 3 times with fresh DMEM, 100 μ L of halouginone diluted in a gradient with 2% FBS-containing DMEM culture solution was added, and the prepared solution of halouginone with the corresponding DMSO concentration was used as a virus control well and placed in a chamber at 37 ℃ for continuous culture 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, 300. mu.L of DAPI was added to each well for staining, the staining was performed for 5min, PBS was rinsed 2 times for 5min each time, and the results were observed with a fluorescence microscope.
The experimental results are shown in fig. 6: a large amount of specific fluorescence was observed in the virus-infected IBRS-2 cells after the virus infection and in the virus-infected group treated with 0.5. mu. mol of Halofuginone, while a small amount of fluorescence was observed in the IBRS-2 cells of the other treated groups. This result further confirms that the anti-foot-and-mouth disease virus activity of Halofuginone on IBRS-2 cells exhibits dose dependence.
2.4 detection of the inhibition of the viral replication phase by FMDV
The IBRS-2 cells were plated in 24-well plates and the culture medium was discarded after the cells grew a monolayer. In the adsorption phase of the virus: FMDV virus solution was added to each well. Then, Halofuginone of different concentrations was added to each well to make the final concentrations 1, 3, 6, 12, 25. mu. mol, and negative control groups without drug were set up, and the cell plates were placed in a refrigerator at 4 ℃ for 1 hour to allow only virus to attach to the cell surface. Discarding virus solution, washing away unbound virus with DMEM, adding 400 μ L culture solution into each well, repeatedly freezing and thawing in-80 deg.C refrigerator for 3 times, collecting virus solution, centrifuging at 10000r/min for 10min to remove cell debris, and collecting supernatant for detecting mRNA expression level. In the virus penetration phase: adding 200 mu L of FMDV virus solution into each hole, putting the cells into an incubator at 37 ℃ for 1h, allowing the viruses to adsorb on the cell surface, washing away the unbound viruses by DMEM, then adding 200 mu L of Halofuginone with different concentrations into each hole, enabling the final concentration of the Halofuginone to be 1, 3, 6, 12 and 25 mu mol respectively, simultaneously establishing a negative control group without adding the drugs, putting the cell plate into an incubator at 37 ℃ for culturing for 1h, removing the drugs, washing away the residual drugs by PBS, adding maintenance solution containing 2 percent of FBS, putting the incubator at 37 ℃ for continuously culturing for 48h, and collecting supernatant to detect the mRNA expression level.
The results of the experiment are shown in FIG. 7. In the virus adsorption (Attachment) stage, the cells are respectively treated with Halofuginone and FMDV virus solution with different concentrations and are acted for 1 hour in an incubator, and the result shows that the mRNA expression level of the virus is not obviously different from that of an untreated group no matter the cells are treated with the Halofuginone with different concentrations, so the Halofuginone does not influence the adsorption of FMDV to the cells. At the viral Entry (Entry) stage, there was no significant difference in mRNA from each drug-treated group compared to the non-drug-treated negative control group, indicating that Halofuginone did not affect the ability of the virus to enter cells.
2.5 evaluation of inhibition time of Halofuginone on foot-and-mouth disease virus infected IBRS-2 cells:
well-grown IBRS-2 cells on a 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 100TCID 50O/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. Halofuginone was added to different wells at 0h, 2h, 4h, 8h, 16h after viral infection to a final concentration of 3. mu. mol. And meanwhile, a negative control without adding the medicine is set. CO at 37 deg.C2Culturing in a constant-temperature cell culture box for 48 h. Different groups of supernatants were collected and q-PCR and Western Blot were used to detect FMDV 2B gene mRNA and FMDVVP1 protein levels, respectively.
The experimental results are shown in FIGS. 8 to 9: treatment of cells with Halofuginone at various time periods after viral infection showed significant inhibition of FMDV mRNA levels (figure 8) and VP1 protein levels (figure 9) compared to negative controls within 0-8 h of FMDV replication. The inhibition of Halofuginone at 16h was not obvious, indicating that Halofuginone only works early in FMDV replication and does not prevent viral replication when it enters late in virus replication.
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 halofuginone in preparation of drugs for preventing foot-and-mouth disease virus infection
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Claims (2)
1. Application of halofuginone in preparing medicine for preventing foot and mouth disease virus infection;
the foot-and-mouth disease virus comprises 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 halofuginone in the medicament is > 1 μmol/L.
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