CN109771419B - Application of AVN-944 in preparation of drugs for preventing foot and mouth disease virus infection - Google Patents
Application of AVN-944 in preparation of drugs for preventing foot and mouth disease virus infection Download PDFInfo
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- CN109771419B CN109771419B CN201910180670.9A CN201910180670A CN109771419B CN 109771419 B CN109771419 B CN 109771419B CN 201910180670 A CN201910180670 A CN 201910180670A CN 109771419 B CN109771419 B CN 109771419B
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Abstract
The invention relates to an application of AVN-944 in preparation of 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 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 drugs, in particular to application of AVN-944 in preparation of drugs 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 AVN-944 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 an application of AVN-944 in preparation of a drug 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 AVN-944 in the medicament is 25-50 mu mol/L.
The invention provides an application of AVN-944 in preparation of a drug for preventing foot-and-mouth disease virus infection. The AVN-944 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 the AVN-944 has low cytotoxicity and has an inhibiting effect on the replication of both A type foot-and-mouth disease virus and O type foot-and-mouth disease virus; further experiments confirmed that AVN-944 only functions early in FMDV replication and does not prevent viral replication when it enters late stage of viral replication. AVN-944 can be used as an effective anti-foot-and-mouth disease virus component.
Drawings
FIG. 1 is a graph of cytotoxicity of different concentrations of AVN-944 on IBRS-2 cells in example 1 of the present invention;
FIG. 2 is a graph showing the inhibitory effect of different concentrations of AVN-944 on type O FMDV infection of IBDS-2 cells in example 1 of the present invention;
FIG. 3 is a graph showing the inhibitory effect of different concentrations of AVN-944 on type A FMDV infection of IBDS-2 cells in example 1 of the present invention;
FIG. 4 is a graph showing the effect of different concentrations of AVN-944 on FMDV type O mRNA inhibition in example 1 of the present invention;
FIG. 5 is a graph showing the effect of different concentrations of AVN-944 on the inhibition of VP1 protein expression in O-type 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 AVN-944 on the inhibition of FMDV protein expression in O-type FMDV infected cells in example 1 of the present invention;
FIG. 7 is a graph showing the effect of AVN-944 on FMDV mRNA inhibition of virus-infected cells at various time periods in example 1 of the present invention;
FIG. 8 is a graph showing the inhibitory effect of AVN-944 on VP1 protein expression in virus-infected cells at different time periods in example 1 of the present invention.
Detailed Description
The invention provides an application of AVN-944 in preparation of a drug 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. AVN-944 has inhibitory effect on cell pathological changes induced by type A foot-and-mouth disease virus and type O foot-and-mouth disease virus, and inhibits virus replication, and AVN-944 only acts in the early stage of FMDV replication, but cannot prevent virus replication in the later stage of virus replication. The source of AVN-944 in the present invention is not particularly limited, and any conventional commercially available product in the art may be used.
In the invention, the concentration of the AVN-944 in the medicament is preferably 25-50 mu mol/L.
The application of the AVN-944 provided by the invention in the preparation of a medicament for preventing foot-and-mouth disease virus infection is further described in detail with reference to the following 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; AVN-944 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 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 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 AVN-944 on IBRS-2 cells:
the cytotoxicity of AVN-944 against IBRS-2 cells was determined by the MTS method. After the cells are paved on a 96-well plate IBRS-2 and fully grow into a monolayer, the upper culture solution of the cells is discarded, the cells are washed for 3 times by using fresh DMEM, AVN-944100 mu L which is diluted by a DMEM culture solution gradient containing 2% FBS is finally added, the corresponding DMSO concentration of the prepared solution of AVN-944 is used as a negative control hole, and the cells are used as cell control holes 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% "the toxicity of AVN-944 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 showed that the cell activity was 95% or more at a concentration of 50. mu. mol or less. When the concentration of the drug was 100. mu. mol, the cell activity decreased rapidly, and the activity rate of the cells was 30%. Therefore, when the anti-FMDV is prepared using this drug, its concentration should not be higher than 50. mu. mol.
2.2 evaluation of AVN-944 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 100TCID50O/MY98/BY/2010 is inoculated. After 1h, virus solution is removed, the cell is washed 3 times by fresh DMEM, AVN-944100 mu L which is diluted by DMEM culture solution containing 2% FBS in a gradient manner is added, the corresponding DMSO concentration of the prepared solution of AVN-944 is used as a virus control hole, and the cell without AVN-944 or 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. 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 AVN-944 at different concentrations was calculated. At the same time, different groups of supernatants were collected, and mRNA of FMDV 2B gene and FMDV VP1 protein levels were detected by q-PCR and Western Blot, 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 annealing at 72 ℃ forAnd stretching for 30 seconds and 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 AVN-944 has an antiviral effect on type A foot-and-mouth disease virus, 100TCID50A/GDMM/CHA/2013 was used to infect cells, and the MTS method was used to determine its antiviral activity.
The experimental results are shown in FIGS. 2 to 5: MTS was used to test whether AVN-944 has antiviral activity against FMDV, and the results showed that AVN-944 provided effective protection of IBDV-2 cells at concentrations greater than 12. mu. mol (FIG. 2) and significantly inhibited the expression of FMDV mRNA levels (FIG. 4) and VP1 protein levels (FIG. 5) when different concentrations of drug were added. Whereas, at concentrations of 12. mu. mol and below, AVN-944 did not provide effective protection of cells. Similarly, when cells were infected with type A foot and mouth disease virus, 6. mu. mol or more of AVN-944 was effective in protecting IBDS-2 cells (FIG. 3), indicating that AVN-944 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, virus solution was removed, washed 3 times with fresh DMEM, and AVN-9441 was added, which was diluted in 2% FBS-containing DMEM medium in a gradient mannermu.L of the suspension was cultured at 37 ℃ for 12 hours in a virus control well containing the corresponding DMSO concentration in the prepared solution of AVN-944. 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 groups treated with IBRS-2 cells after virus infection and AVN-944 at concentrations of 3, 6 and 12. mu. mol, whereas a small amount of fluorescence was observed in IBRS-2 cells of the other groups. This result further confirms that AVN-944 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 AVN-944:
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 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. AVN-944 was added to different wells at 0h, 2h, 4h, 8h, 16h after viral infection to a final concentration of 50. 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 FMDV VP1 protein levels, respectively.
The experimental results are shown in FIGS. 7 to 8: cells were treated with AVN-944 at various 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 inhibitory effect of AVN-944 at 16h was not evident, indicating that AVN-944 only acts early in FMDV replication and fails to prevent viral replication when it enters late stage of viral 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 AVN-944 in preparation of drugs for preventing foot-and-mouth disease virus infection
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Claims (1)
- The application of AVN-944 in preparing medicine for preventing foot and mouth disease virus infection; the foot-and-mouth disease virus is A type foot-and-mouth disease virus and O type foot-and-mouth disease virus; the concentration of the AVN-944 in the medicine is 25-50 mu mol/L.
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Non-Patent Citations (5)
| Title |
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| Curing of foot-and-mouth disease virus from persistently infected cells by ribavirin involves enhanced mutagenesis;Airaksinen等;《Virology》;20030705;第311卷(第2期);第339-349页 * |
| Identification of Inhibitors of ZIKV Replication;Desarey Morales Vasquez等;《Viruses》;20200918;全文 * |
| Lifecycle modelling systems support inosine monophosphate dehydrogenase (IMPDH) as a pro-viral factor and antiviral target for New World arenaviruses;Hoenen, Thomas等;《Antiviral Research》;20180930;第157卷;第140-150页 * |
| Synthesis and antiviral activity of a novel class of (5-oxazolyl)phenyl amines;Zhao-Jin Zhong等;《European Journal of Medicinal Chemistry》;20130815;第32-43页 * |
| 以次黄嘌呤核苷酸脱氢酶为靶点的药物研发;张大军等;《中国医药生物技术》;20120430;第7卷(第2期);第130-135页 * |
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