Application of muscone in preparation of virus inhibitor

Abstract

The invention relates to the field of biological medicine, in particular to application of muscone in preparation of a virus inhibitor. The virus inhibitor is used for inhibiting replication of Japanese encephalitis virus and / or porcine epidemic diarrhea virus. It is found that muscone has no cytotoxicity, does not directly inactivate viruses, generates an antiviral effect on Japanese encephalitis viruses by inhibiting signal channels such as NLRP3 / Caspase-1, Caspase-9 / Caspase-3 / Bax / BCL-2, JNK / ERK / P38 and the like, and inhibits porcine epidemic diarrhea viruses from invading cells by reducing the level of litter protein. The antiviral effect is obvious, the action mechanism is clear, the selectivity is high, and the safety is better.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to an application of muscone in preparation of a virus inhibitor. BACKGROUND

[0002] Japanese encephalitis and porcine epidemic diarrhea virus are important pathogens that endanger the pig industry, and their high infectivity and high mortality to pig populations make them the focus of prevention and control.

[0003] Japanese encephalitis, also known as epidemic encephalitis B (hereinafter referred to as encephalitis B), is a zoonosis caused by Japanese encephalitis virus (JEV). JEV belongs to the Flaviviridae family and is a neurotropic virus. The main target cell of infection is the neuron cell. Both direct infection of the virus and immune inflammatory reaction mediated by the virus can cause neuron cell death. The proliferation titer of the virus in the periphery is the key to determine whether JEV can break through the blood-brain barrier and enter the central nervous system. Therefore, controlling the proliferation of the virus is crucial for intervention of encephalitis B progression. At present, the pathogenesis of encephalitis B has not been fully elucidated, and there is no effective antiviral drug for the treatment of encephalitis B. The clinical treatment is mainly symptomatic and supportive treatment. Encephalitis B is a major epidemic disease that causes serious harm to the pig breeding industry, mainly affecting the reproductive performance of sows, such as abortion, stillbirth and mummified fetus in pregnant sows, and orchitis in boars, which can cause persistent high fever in adult pigs and growing pigs, leading to encephalitis in newborn piglets.

[0004] The pathogen of porcine epidemic diarrhea is porcine epidemic diarrhea virus (PEDV), which belongs to the coronavirus. At present, PEDV has become one of the key pathogens that restrict the steady development of China's pig industry, and its influence is only second to African swine fever virus and porcine reproductive and respiratory syndrome virus.

[0005] The above-mentioned viruses have the problems of rapid spread, high mortality, great harm to the pig industry, and poor treatment effect of existing drugs, so it is a technical problem in the field of drug cost to find a new drug with significant curative effect. SUMMARY

[0006] In order to solve the above problems, the application provides a use of muscone in preparation of a virus inhibitor, which can provide a reference for the current pig industry prevention and control scheme.

[0007] In order to achieve the above purpose, the application provides the following technical scheme: In a first aspect, the application provides a use of muscone in preparation of a virus inhibitor for inhibiting the replication of Japanese encephalitis virus and / or porcine epidemic diarrhea virus.

[0008] In some embodiments, the viral inhibitor inhibits the replication of Japanese encephalitis virus by regulating at least one of NLRP3 / Caspase-1 signaling pathway, Caspase-9 / Caspase-3 / Bax / Bcl-2 signaling pathway, JNK / ERK / P38 signaling pathway.

[0009] In some embodiments, the viral inhibitor is capable of down-regulating the protein level of at least one of NLRP3, Caspase-1, p-JNL, Cleaved Caspase-9, Cleaved Caspase-3, Bax; and / or the viral inhibitor is capable of up-regulating the protein level of Bcl-2.

[0010] In some embodiments, the viral inhibitor is a Japanese encephalitis virus inhibitor that inhibits Caspase-9 / Caspase-3 / Bax / Bcl-2 signaling pathway; or the viral inhibitor is a Japanese encephalitis virus inhibitor that inhibits NLRP3 / Caspase-1 signaling pathway; or the viral inhibitor is a Japanese encephalitis virus inhibitor that inhibits JNK / ERK / P38 signaling pathway.

[0011] In some embodiments, the viral inhibitor inhibits the replication of porcine epidemic diarrhea virus by down-regulating the level of Caveolin-1 protein; and / or the viral inhibitor inhibits the replication of porcine epidemic diarrhea virus by inhibiting viral invasion into cells.

[0012] In some embodiments, the viral inhibitor is a porcine epidemic diarrhea virus inhibitor that inhibits the expression of Caveolin-1 protein.

[0013] In some embodiments, the viral inhibitor is a drug for preventing, diagnosing and treating Japanese encephalitis and / or porcine epidemic diarrhea.

[0014] In some embodiments, the viral inhibitor is used to prepare at least one of a pharmaceutical product, an animal foodstuff and additives thereof, a bait, a cosmetic product, a perfume.

[0015] In a second aspect, the present application further provides a viral inhibitor, the viral inhibitor comprising: a first active ingredient, the first active ingredient comprising: muscone, the viral inhibitor being used for inhibiting the replication of Japanese encephalitis virus and / or porcine epidemic diarrhea virus.

[0016] In some embodiments, the viral inhibitor further comprises one of an adjuvant, a carrier, and a second active ingredient capable of increasing the activity of the first active ingredient or reducing the adverse reaction of the first active ingredient; and / or The viral inhibitor is used for preventing, diagnosing and treating Japanese encephalitis and / or porcine epidemic diarrhea.

[0017] Experiments have verified that muscone has no cytotoxicity to BHK-21, HMC3, SK-N-SH, Vero-81 and other cells, and cannot directly inactivate Japanese encephalitis virus and porcine epidemic diarrhea virus, and muscone plays a viral inhibitory role by inhibiting viral protein expression to inhibit viral replication. Further, muscone produces an antiviral effect on Japanese encephalitis virus by inhibiting NLRP3 / Caspase-1, Caspase-9 / Caspase-3 / Bax / BCL-2, JNK / ERK / P38 and other signaling pathways, and inhibits the invasion of porcine epidemic diarrhea virus into cells by reducing caveolin levels.

[0018] In addition, muscone not only has a brand-new antiviral mechanism, but also has a very significant antiviral effect on JEV at a concentration of 10 μM, that is, it can exhibit a very strong antiviral effect. The antiviral effect of muscone on PEDV is very significant at a concentration of 20 μM, that is, it can exhibit a very strong anti-porcine epidemic diarrhea virus infection effect.

[0019] In addition, muscone is used for an antiviral drug with low residue, no pollution, easy to be absorbed by an animal body, high biological metabolism rate and no pollution in excretion.

[0020] Therefore, muscone has a significant antiviral effect on Japanese encephalitis virus and porcine epidemic diarrhea virus, a clear mechanism of action, high selectivity and good safety. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below.

[0022] Figure 1 It is the cell toxicity detection result of muscone, wherein A is BHK-21, B is SK-N-SH, C is HMC3, and D is Vero-81.

[0023] Figure 2 It is the virus inactivation performance test result of muscone on JEV, and ns represents no significant difference.

[0024] Figure 3 It is the viral replication inhibition performance test result of muscone on JEV, A is BHK-21+JEV, B is SK-N-SH+JEV, and C is HMC3+JEV.

[0025] Figure 4 The research results of muscone on the inhibition mechanism of JEV from the NLRP3 / Caspase-1 signal pathway.

[0026] Figure 5 The research results of muscone on the inhibition mechanism of JEV from the JNK / ERK / P38 signal pathway.

[0027] Figure 6 The research results of muscone on the inhibition mechanism of JEV from the Caspase-9 / Caspase-3 / Bax / Bcl-2 signal pathway.

[0028] Figure 7 The test results of the virus inactivation performance of muscone on PEDV, ns represents no significant difference.

[0029] Figure 8 The test results of the replication inhibition performance of muscone on PEDV.

[0030] Figure 9 The test results of the degradation effect of muscone on caveolin.

[0031] Figure 10 The test results of the inhibition of muscone on the replication of EGFP-PEDV.

[0032] Figure 11 The experimental procedure of Example 4.

[0033] Figure 12 The statistical results of clinical indexes of Example 4.

[0034] Figure 13 The statistical results of survival rates of Example 4.

[0035] Figure 14 The detection results of neuroinflammation indexes of Example 4.

[0036] Figure 15 The expression of interferon stimulated genes IFIT1, Mx1 and Mx2 of Example 4.

[0037] Figure 16 The expression of JEV-C gene of Example 4.

[0038] Figure 17 The detection results of the virus titers of JEV in the brain, liver, spleen and heart tissues of mice of Example 4. DETAILED DESCRIPTION

[0039] In order to further illustrate the present application, the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of the present application. Muscone

[0040] Muscone is one of the active ingredients extracted from the dry secretion musk of mature male Moschus berezovskii Flerov or Moschus moschiferus L, which is the main fragrance component of musk. The chemical name of muscone is 3-methyl pentadecanone, the molecular formula is C 16 H 30 O, the molecular weight is 238.42, which is a yellowish oily liquid with a special fragrance, slightly soluble in water, and can be mixed with ethanol. The molecular structure is as follows: Japanese encephalitis virus

[0041] Japanese encephalitis virus (JEV) belongs to the family Flaviviridae and is a neurotropic virus. The main target cells of JEV infection are neurons. JEV infection directly and through immune inflammatory response mediated by JEV can cause neuronal cell death, leading to Japanese encephalitis (Japanese encephalitis, JE).

[0042] JEV is an enveloped virus, and the virus particle is about 35-40 nm in size. Its genome is a single-stranded positive-sense RNA with a total length of about 11 kb. The genomic structure includes: 5' untranslated region (5'UTR), about 95 nucleotides, containing a type I cap structure , involved in the regulation of translation initiation; open reading frame (ORF), encoding a polyprotein (about 10,299 nucleotides), which is cleaved into 10 mature proteins by host and viral proteases; 3' untranslated region (3'UTR): about 574-585 nucleotides, without poly(A) tail, can form a conserved secondary structure (such as stem-loop structure), which is related to viral replication and virulence. The genomic RNA encodes a polyprotein on the endoplasmic reticulum (ER) membrane, which is cleaved into three structural proteins (Capsid, prM, and Envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Based on the sequence of the E gene, JEV is divided into five genotypes (GI-GV). The main epidemic types in China are GI and GIII, and the replication efficiency and pathogenicity of GI type are higher than those of GIII type, which may be related to the differences in amino acids at positions 55 (D / E) and 65 (E / D) of NS2B protein. These sites affect the activity of the protease. JEV has only one serotype, and there is cross-immune protection between genotypes.

[0043] Porcine epidemic diarrhea virus Porcine Epidemic Diarrhea Virus (PEDV) is the main pathogen causing epidemic diarrhea in pigs, which belongs to the Coronaviridae family and is highly harmful to piglets, especially newborn piglets. It is one of the important viral infectious disease pathogens in the global pig industry. PEDV is a single-stranded positive-sense RNA virus, and the virion is spherical or oval with a membrane, and the surface is covered with rod-shaped spines (spike protein, S protein), which is closely related to the host specificity, pathogenicity and immunogenicity of the virus. S protein is the key antigen for inducing the production of neutralizing antibodies and is the main target for vaccine development. PEDV mainly infects pigs through the oral-canal route, and the virus enters the small intestine epithelial cells (especially the villus epithelial cells of the duodenum and jejunum) through specific recognition and binding to the receptors (such as aminopeptidase N) on the surface of the cells, and then invades the cells and replicates in them. Virus replication can cause small intestinal villus epithelial cells to degenerate, necrosis, and shed, making the small intestinal villi short, thick, or even fused, severely damaging the absorption function of the intestinal tract, leading to water and electrolyte absorption disorders in the intestinal tract, causing severe diarrhea and dehydration, which is the core mechanism of PEDV pathogenesis.

[0044] In order to more clearly illustrate the present application, the present application will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the specific description below is illustrative rather than limiting, and should not limit the scope of protection of the present application.

[0045] The experimental materials used in the following examples include but are not limited to: Porcine Epidemic Diarrhea Virus (PEDV), Japanese Encephalitis Virus (JEV), and BHK-21 (hamster kidney fibroblast cells), HMC3 (human microglial cells), and Vero-81 (African green monkey kidney cells) were all from the laboratory group.

[0046] CCK-8 kit was purchased from Wuhan Sevyl Biotech Co., Ltd.

[0047] Muscone (CAS: 541-91-3) was purchased from Selleck Company. Example 1 Cell toxicity experiment of muscone

[0048] BHK-21, HMC3, SK-N-SH, Vero-81 and other cells were seeded into 96-well plates at a density of about 80%. After 3 PBS washes, the cells were cultured in muscone at concentrations of 2 µM, 10 µM, 20 µM, 50 µM and 100 µM. DMSO-treated cells were used as controls. After 24 h, the cells were washed and OD value determination was performed at 450 nm using a microplate reader according to the manufacturer's instructions. Cell toxicity was expressed by the formula: cell survival rate (%) = (OD experimental well - OD blank well) / (OD control well - OD blank well) x 100%. Each group of experiments was set in triplicate, and the data were statistically analyzed as mean (Mean) ± standard deviation (SD).

[0049] As shown in Figure 1 , there was no significant difference in cell activity in the experimental groups treated with different concentrations of muscone within a certain drug concentration range (e.g., less than or equal to 20 µM), indicating that muscone less than or equal to 20 µM had no significant cytotoxicity to BHK-21, HMC3, SK-N-SH, Vero-81 and other cells. Example 2 Study of muscone on JEV

[0050] 1. Test of the virus inactivation performance of muscone on JEV JEV virus solution was mixed with different concentrations of muscone, with a total volume of 1 mL, and incubated at 37°C for 2 h at increasing concentrations. Then the mixture was added to cells, which were then incubated at 37°C for another 2 h, and unbound viruses were removed by PBS washing. The control group of cells was infected with the same amount of JEV without muscone at a concentration of 0. The cells were incubated at 37°C, and after 24 h, RNA samples were collected and reverse-transcribed into cDNA, and q-PCR was used to detect the copy number of JEV genome. When the results were statistically analyzed, the change fold of JEV mRNA level in the control group with muscone concentration of 0 was 1, and the JEV genome copy number data of other experimental groups were calculated.

[0051] As shown in Figure 2 , muscone had no direct inactivation effect on JEV at a concentration of 0-20 µM, confirming that muscone inhibited the activity of JEV without direct inactivation of the virus.

[0052] 2. Test of the replication inhibition performance of muscone on JEV BHK-21, SK-N-SH and HMC3 cells were respectively seeded into 6-well plates at a density of 1×10 5Cells were seeded at a density of 100 cells / well into 12-well plates. After reaching approximately 80% cell density, the cells were washed twice with serum-free DMEM, seeded with JEVs, and incubated at 37°C for 2 hours. Cell maintenance medium of different concentrations of thymol was then added. Cell samples were collected after 24 hours and subjected to Western blotting. The control group (represented as "con" in the figure) was not seeded with JEVs, and thymol was replaced with DMSO.

[0053] The results are as follows Figure 3 As shown, compared with the experimental group with a muscone concentration of 0 μM, the expression level of JEV NS5 in cells treated with different concentrations of muscone was significantly reduced, proving that muscone can significantly inhibit the replication of JEV virus.

[0054] 3. Study on the inhibitory mechanism of muscone on JEV (1) NLRP3 / Caspase-1 signaling pathway With 1×10 5 HMC3 cells were seeded into 12-well plates at a density of approximately 80%. After washing twice with serum-free DMEM, JEVs were inoculated and cultured at 37°C for 2 hours. Cell maintenance medium containing 0-20 μM thymol was then added. Cell samples were collected after 24 hours for Western blot analysis. The control group (represented as "con" in the figure) was not seeded with JEVs, and thymol was replaced with DMSO.

[0055] The results are as follows Figure 4 As shown, JEV infection significantly activates the NLRP3 / Caspase-1 pathway 24 hours after infection. Compared with the untreated group, the NLRP3 / Caspase-1 protein level decreased in the 20 μM muscone treatment group, indicating that muscone significantly inhibits the activation of the NLRP3 / Caspase-1 pathway.

[0056] (2) JNK / ERK / P38 signal path With 1×10 5 HMC3 cells were seeded into 12-well plates at a density of approximately 80%. After washing twice with serum-free DMEM, JEVs were inoculated and cultured at 37°C for 2 hours. Cell maintenance medium containing 0-20 μM thymol was then added. Cell samples were collected after 24 hours for Western blot analysis. The control group (represented as "con" in the figure) was not seeded with JEVs, and thymol was replaced with DMSO.

[0057] The results are as follows Figure 5As shown, JEV infection significantly activates the JNK / ERK / P38 pathway 24 hours later. Compared with the untreated group, the p-JNL protein level decreased in the 20 μM muscone treatment group, indicating that muscone significantly inhibits the activation of the JNK / ERK / P38 pathway.

[0058] (3) Caspase-9 / Caspase-3 / Bax / Bcl-2 signaling pathway With 2×10 5 SK-N-SH cells were seeded into 12-well plates at a density of 10 cells / well. After reaching approximately 80% cell density, the cells were washed twice with serum-free DMEM, inoculated with JEVs, and cultured at 37°C for 2 hours. Cell maintenance medium containing 0-20 μM thymol was then added. Cell samples were collected after 24 hours for Western blot analysis. The control group (represented as "con" in the figure) was not seeded with JEVs, and thymol was replaced with DMSO.

[0059] The results are as follows Figure 6 As shown, the Caspase-9 / Caspase-3 / Bax / Bcl-2 pathway was significantly activated 24 hours after JEV infection. Compared with the untreated group, the 20 μM muscone treatment group showed decreased levels of leached Caspase-9, leached Caspase-3, and Bax proteins, and increased levels of Bcl-2 protein, indicating that muscone significantly inhibited the activation of the Caspase-9 / Caspase-3 / Bax / Bcl-2 pathway. Example 3: Study on muscone against PEDV

[0060] 1. Test of the viral inactivation activity of muscone against PEDV PEDV virus solution was mixed with different concentrations of muscone, with a total volume of 1 mL, and incubated at 37°C for 2 h in increments of concentration. The mixture was then inoculated into cells, followed by incubation at 37°C for another 2 h. Unbound virus was removed by washing with PBS. Control cells were infected with an equal amount of JEV at a muscone concentration of 0. Cells were incubated at 37°C until more than 80% CPE was formed, and the supernatant was collected after freeze-thaw cycles for testing. Virus inactivation was subsequently assessed using TCID50.

[0061] The results are as follows Figure 7 As shown, muscone did not directly inactivate PEDV at concentrations of 0-20 μM, confirming that muscone inhibits PEDV activity not through direct viral inactivation.

[0062] 2. Test on the inhibitory effect of muscone on PEDV replication Vero-81 cells were loaded at a rate of 1×10⁻⁶. 5Cells were seeded at a density of 100 cells / well into 12-well plates. After reaching a density of approximately 80%, the cells were washed twice with serum-free DMEM, seeded with PEDV, and incubated at 37°C for 2 hours. The liquid in the wells was discarded, and cell maintenance medium of different concentrations of muscone was added. Cell samples were collected after 24 hours and subjected to Western blotting. The control group (represented as "con" in the figure) had a muscone concentration of 0 μM.

[0063] The results are as follows Figure 8 As shown, compared with the control group with a 0 μM musketone concentration, the expression level of PEDV-N in cells treated with different concentrations of musketone was significantly reduced, demonstrating that musketone can significantly inhibit the replication of PEDV virus.

[0064] 3. Study on the inhibitory mechanism of muscone on PEDV (1) Muscone can degrade caveolin With 1×10 5 Vero-81 cells were seeded into 12-well plates at a density of approximately 80%. After washing twice with serum-free DMEM, cells were inoculated with PEDV and cultured at 37°C for 2 hours. The liquid in the wells was discarded, and cell maintenance medium containing 0-20 μM thymol was added. Cell samples were collected after 24 hours for Western blot analysis. The control group (represented as "con" in the figure) was either inoculated with PEDV (represented as "+") or not inoculated with PEDV (represented as "-"), and thymol was replaced with DMSO.

[0065] Caveolin-1 is a type of structural protein on the membrane of host cells (such as small intestinal epithelial cells of pigs). Its main functions include participating in cell signal transduction, endocytosis (such as cholesterol transport), and maintaining the structure of the cell membrane.

[0066] The results are as follows Figure 9 As shown, caveolin-1 protein was significantly degraded under the action of muscone. Compared with the untreated group, the caveolin-1 protein level decreased in the 20 μM muscone-treated group, indicating that muscone can degrade caveolin-1 at the protein level, thereby reducing the expression level of caveolin-1.

[0067] (2) Muscone inhibits PEDV invasion of cells by degrading caveolin. The cell suspension was added to the cell climbing sheet placed in a 12-well cell culture plate, and when the Vero-81 cell density reached 60%, the cells were washed with PBS, and 2 μM, 10 μM, and 20 μM muscone were used to pretreat the Vero-81 cells at 37°C for 1 h. The supernatant was discarded, the cells were washed with PBS, and EGFP-overexpressing PEDV (EGFP-PEDV, MOI 0.05) was added to incubate the cells for 2 h. EGFP is green fluorescent protein. The cells were washed again and treated with different concentrations of muscone for 24 h. The supernatant was discarded, and the cells were fixed with 4% polyformaldehyde at room temperature for 10 min and washed with PBS. The cells were blocked with 1% BSA at room temperature for 30 min. Caveolin-1 monoclonal rabbit antibody was incubated at 4°C overnight. Then the antibody was discarded, and the cells were washed with PBS. Goat anti-rabbit IgG labeled with Cy3 was incubated at room temperature for 1 h in the dark, and the cells were washed with PBS. Nuclei were stained with DAPI at room temperature for 10 min in the dark, and the cells were washed with PBS. 1 mL of PBS was added to the well, and the pictures were taken and processed using an OL YMPUS laser scanning confocal microscope. Among them, the negative control group and the blank control group were set, and compared with the above experimental groups, the negative control group (indicated as: CON) was not treated with muscone after the EGFP-PEDV was added, and the blank control group (indicated as: Mock) was not added with EGFP-PEDV after the muscone was pretreated, but the cells were then treated with muscone.

[0068] The results are shown in Figure 10 By observing the fluorescence, it can be found that the EGFP fluorescence becomes weaker with the increase of the concentration of muscone, indicating that muscone can inhibit the replication of EGFP-PEDV, and inhibit the replication of PEDV in a dose-dependent manner. Figure 9 The results presented: muscone significantly degrades the expression of Caveolin-1. Since the main function of Caveolin-1 is to participate in cell signal transduction, material endocytosis (such as cholesterol transport), and maintenance of cell membrane structure, it is speculated that muscone inhibits the invasion of PEDV into cells by degrading caveolin, thereby inhibiting the replication of EGFP-PEDV. Example 4 Animal experiment

[0069] Protective effect of muscone on JEV-infected mice (1) Take 40 C57 mice of 4-6 weeks old, and randomly divide them into 4 groups, including: blank control group, single drug control group, JEV group, JEV+drug group, 10 mice in each group. Among them, the blank control group is represented as: DMSO+DMEM (also represented as DMSO), which means that DMSO is given when the drug is given, and DMEM is given when the virus is inoculated. The single drug control group is represented as: Muscone+DMEM (also represented as Muscone), which means that Muscone is given when the drug is given, and DMEM is given when the virus is inoculated. The JEV group is represented as: DMSO+JEV (also represented as JEV), which means that DMSO is given when the drug is given, and JEV is given when the virus is inoculated. The JEV+drug group is represented as: Muscone+JEV, which means that Muscone is given when the drug is given, and JEV is given when the virus is inoculated. Specifically, the dose of Muscone is 12 mg / kg per day, the dose of DMEM is 100 μL per day, and the virus titer of JEV is 2×10 6 PFU / mL, and the inoculation amount is 100 μL.

[0070] The specific experimental procedure is shown in Figure 11 The virus is inoculated on the first day, and the drug is given by intraperitoneal injection on the 3rd, 4th, and 5th days, 12 mg / kg per day, and the mice are observed every day after inoculation, and the behavior, body weight change, and death of the mice are recorded.

[0071] On the 6th day after inoculation, 5 mice were randomly selected from each group, and their brain tissues and important organs were taken, a part of which was homogenized, and RNA and protein samples were extracted, and the homogenate supernatant was collected, and the expression of (pro) inflammatory factors and interferon was detected by fluorescent quantitative RT-PCR, and the virus titer was detected by plaque assay, and the remaining 5 mice were continuously observed until the 21st day after inoculation to record the body weight change and survival, and the survival curve was drawn.

[0072] The body weight change, state performance, and survival recorded every day were summarized, and the clinical index and survival rate of each group were counted.

[0073] Figure 12 The clinical index statistical results are shown in the figure, 60% of the mice in the JEV group showed clinical symptoms on the 5th day after inoculation; the Muscone+JEV group had fewer symptoms than the DMSO+JEV group, and even no clinical symptoms appeared. On the 8th day, all the mice in the Muscone+JEV group showed clinical symptoms, but only 20% showed clinical symptoms. On the 9th day, all the mice in the JEV group died, and 20% of the mice in the Muscone+JEV group showed symptoms compared with the 8th day. Until the 15th day, 50% of the mice in the Muscone+JEV group showed symptoms, and the remaining 50% of the mice did not show symptoms until the 21st day. It is shown that muscone can reduce the clinical symptoms of mice after JEV infection.

[0074] Figure 13 The survival rate statistics are shown in the figure. Mice in the JEV group began dying on day 6, while the blank control group (…) Figure 13 The Chinese text refers to DMSO and the control group treated alone (DMSO). Figure 13 Mice treated with muscone (represented as 'Muscone') survived until the end of the experiment. In the muscone+JEV group, 20% of the mice died on day 8, with a mortality rate of 50% by the end of the experiment. All mice in the JEV group died on day 9. These results indicate that muscone treatment can improve the survival rate of mice infected with JEV.

[0075] (2) Muscone can inhibit neuroinflammation induced by JEV infection in mice.

[0076] Five groups of mouse brain tissue were randomly selected, homogenized, and RNA was extracted. The expression of pro-inflammatory cytokines TNF-α, IL-1β, and CCL-5 was detected by quantitative real-time RT-PCR. like Figure 14 As shown, in mouse brain tissue, the expression levels of TNF-α, IL-1β, and CCL-5 genes in experimental group 1 were significantly lower than those in the JEV group, indicating that muscone can inhibit neuroinflammation induced by JEV infection in mice.

[0077] (3) Muscone can inhibit JEV replication in mice by upregulating the transcription level of interferon-stimulated genes.

[0078] Five groups of mouse brain tissue were randomly selected, homogenized, and RNA was extracted. The expression of interferon-stimulated genes IFIT1, Mx1, and Mx2 was detected by quantitative real-time RT-PCR.

[0079] like Figure 15 As shown, in mouse brain tissue, the expression levels of TIFIT1, Mx1, and Mx2 in the Muscone+JEV group were significantly higher than those in the JEV group; Figure 16 As shown, the expression level of the JEV-C gene in experimental group 2 was significantly lower than that in the JEV group, indicating that muscone can inhibit the replication of JEV in mice by upregulating the transcription level of interferon-stimulated genes.

[0080] (4) The viral load in the brain and peripheral blood of mice in the muscone group was significantly reduced. Brain, liver, spleen, and heart tissues were aseptically collected from mice. 0.3g of tissue was added to 1mL of PBS and homogenized in a cryogenic homogenizer. The supernatant was collected. The supernatant was filtered through a 0.22μm filter under aseptic conditions. The filtered homogenate was then subjected to TCID45. 50 The test is performed to determine the amount of virus present. Simply put, it involves diluting the product by 10... −1 Up to 10 −8Supernatant samples were added to BHK-21 cells at 10-fold dilutions. 96-well plates were placed for 4 to 5 days. Each dilution was added to 4 to 8 wells. Cytopathic effect (CPE) was observed under light microscope and TCID50 was calculated by Reed-Muench and Karber 50 .

[0081] As shown in Figure 17 , the viral titers of JEV in the brain, liver, spleen and heart tissues of the mice in the Muscone+JEV group were significantly lower than those in the JEV group, indicating that muscone can reduce the viral load of JEV in the brain and periphery of the mice after JEV infection.

[0082] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. Use of muscone in the preparation of a virus inhibitor for inhibiting the replication of Japanese encephalitis virus and / or porcine epidemic diarrhea virus.

2. The use according to claim 1, characterized in that, The viral inhibitor inhibits the replication of Japanese encephalitis virus by regulating at least one of the following signaling pathways: Caspase-9 / Caspase-3 / Bax / Bcl-2, NLRP3 / Caspase-1, and JNK / ERK / P38.

3. The use according to claim 1, characterized in that, The viral inhibitor can downregulate the protein levels of at least one of NLRP3, Caspase-1, p-JNL, Cleaved Caspase-9, Cleaved Caspase-3, and Bax; and / or The viral inhibitor can upregulate the protein level of Bcl-2.

4. The use according to claim 1, characterized in that, The viral inhibitor is a Japanese encephalitis virus inhibitor that inhibits the Caspase-9 / Caspase-3 / Bax / Bcl-2 signaling pathway; or The viral inhibitor is a Japanese encephalitis virus inhibitor that inhibits the NLRP3 / Caspase-1 signaling pathway; or The viral inhibitor is a Japanese encephalitis virus inhibitor that inhibits the JNK / ERK / P38 signaling pathway.

5. The use according to claim 1, characterized in that, The viral inhibitor suppresses the replication of porcine epidemic diarrhea virus by downregulating the level of Caveolin-1 protein; and / or The viral inhibitor suppresses the replication of porcine epidemic diarrhea virus by inhibiting viral invasion of cells.

6. The use according to claim 1, characterized in that, The virus inhibitor is a porcine epidemic diarrhea virus inhibitor that inhibits Caveolin-1 protein expression.

7. The use according to any one of claims 1 to 6, characterized in that, The viral inhibitor is a drug for the prevention, diagnosis and treatment of Japanese encephalitis and / or swine epidemic diarrhea.

8. The use according to any one of claims 1 to 6, characterized in that, The virus inhibitor is used to prepare at least one of the following: pharmaceuticals, animal foods and their additives, feed, cosmetics, and perfumes.

9. A viral inhibitor, characterized in that, The virus inhibitor comprises a first active ingredient, which includes muscone, and is used to inhibit the replication of Japanese encephalitis virus and / or porcine epidemic diarrhea virus.

10. The virus inhibitor according to claim 9, characterized in that, The virus inhibitor further includes: one of an excipient, a carrier, and a second active ingredient, wherein the second active ingredient can enhance the activity of the first active ingredient or reduce the adverse reactions of the first active ingredient; and / or The viral inhibitors are used to prevent, diagnose, and treat Japanese encephalitis and / or swine epidemic diarrhea.

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