The molecular weight of Thaflavanin-3apos is 10,000-10,000; application of-Gallate or NAD < + > in preparation of product for preventing or treating monkey pox virus infection

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of Thaflavanin-3 '-Gallate or NAD < + > in preparation of a product for preventing or treating monkey pox virus infection. The invention finds that the protein OPG188 of the monkey pox virus can significantly inhibit the natural immune process of the monkey pox virus, compounds Thaflavanin-3 '-Gallate and NAD + which have the highest affinity with the OPG188 are screened out through molecular virtual docking, and the action effects of the compounds are verified through cell experiments and molecular experiments, which indicates that the Thaflavanin-3'-Gallate and NAD + have the effect of inhibiting the replication of the monkey pox virus. The compound can be used for preparing medicines for resisting monkey pox virus infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of Theaflavin-3'-Gallate or NAD+ in preparation of products for preventing or treating monkeypox virus infection. BACKGROUND

[0002] Monkeypox virus (MPXV) as a member of orthopoxvirus genus belongs to double-stranded DNA enveloped virus together with variola major virus (VARV) and cowpox virus (CPXV). The virus is transmitted through respiratory tract and contact, and can cause fever, rash, lymphadenopathy and other symptoms, and even organ failure and death in severe cases, which poses a significant threat to human health. The monkeypox epidemic has been upgraded to a global public health emergency.

[0003] At present, the approved drugs for orthopoxvirus infection are only brincidofovir (BCV) and tecovirimat, in addition, new drugs can also be applied for monkeypox treatment through emergency investigation of new drugs (EIND). However, the efficacy of tecovirimat in clinical trials is not ideal, and the main reason may be that it mainly targets the conserved target of orthopoxvirus (such as viral envelope protein), without fully considering the unique pathogenic mechanism of MPXV, resulting in insufficient specificity for monkeypox.

[0004] Host innate immunity is the first line of defense against viruses and controls viral replication in the early stage of viral infection. Studies have shown that MPXV can escape the clearance of host innate immunity by inhibiting the production of type I interferon. Its main mechanism is to activate the downstream signaling pathway by recognizing viral nucleic acid (DNA or RNA), initiate the expression of type I interferon and inflammatory cytokines, and induce cell death, thereby exerting antiviral effect. However, the mechanism of activating innate immunity and inducing expression of type I interferon after MPXV infection of host cells is not clear.

[0005] In view of the high pathogenicity of monkeypox virus, the limitations of existing drugs, and the research gap in viral immune escape mechanism, it is an urgent technical problem to develop high-efficiency inhibitors targeting specific targets of MPXV. SUMMARY

[0006] In view of the above problems, the purpose of the present application is to provide the application of Theaflavin-3'-Gallate or NAD+ in preparation of products for preventing or treating monkeypox virus infection.

[0007] The purpose of the present application can be realized by the following technical solutions: In a first aspect of the present application, there is provided a use of Theaflavin-3'-Gallate or NAD+ in the preparation of a product for preventing or treating monkeypox virus infection.

[0008] In a second aspect of the present application, there is provided a method for inhibiting monkeypox virus infection for non-therapeutic purposes in vitro, comprising administering an effective dose of Theaflavin-3'-Gallate or NAD+ to an infected system or an infected individual.

[0009] The above technical solution has the following beneficial effects relative to the prior art: The present application has found that monkeypox virus can inhibit the host's natural immune process against viruses by expressing OPG188. Through large-scale molecular virtual docking, the virtual screening docking region takes the active site of Poxin in the OPG188 protein as the center, screens candidate compounds that may interact with the OPG188 protein, and further screens the compound Theaflavin-3'-Gallate and NAD+ with the highest affinity to OPG188 through candidate gene experiments. The effect of Theaflavin-3'-Gallate and NAD+ is verified through cell experiments and molecular experiments, indicating that Theaflavin-3'-Gallate and NAD+ have the effect of inhibiting MPXV replication and can be used for preparing an anti-monkeypox virus infection drug. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The results of the reporter gene experiment for the 17 compounds screened in the embodiments of the present application are shown in the schematic diagram, wherein ** indicates p<0.01, and *** indicates p<0.001; Figure 2 The molecular docking of the small molecule compounds NAD+ and Theaflavin-3'-Gallate with the OPG188 protein is shown in the schematic diagram, wherein A is the chemical structural formula of Theaflavin-3'-Gallate, B is the chemical structural formula of NAD+, C is the molecular docking of Theaflavin-3'-Gallate with OPG188, which shows that it occupies the catalytic active site of OPG188, and D is the molecular docking of NAD+ with OPG188, which shows that it occupies the catalytic active site of OPG188; Figure 3 The detection results of the expression of type I interferon IFNB1 and antiviral genes ISG54 / ISG56 in the embodiments of the present application are shown in the schematic diagram, wherein A is the detection result of the expression of type I interferon IFNB1, B is the detection result of the expression of the antiviral gene ISG54, and C is the detection result of the expression of the antiviral gene ISG56; Figure 4Theaflavin-3'-Gallate and NAD+ on monkeypox virus in THP-1 cells; wherein, A is a schematic diagram of monkeypox virus OPG187 gene expression, B is a schematic diagram of monkeypox virus OPG188 gene expression, C is a schematic diagram of monkeypox virus OPG189 gene expression; Figure 5 Theaflavin-3'-Gallate and NAD+ on THP-1 cell toxicity determination schematic diagram; wherein, A is a schematic diagram of Theaflavin-3'-Gallate on THP-1 cell toxicity determination, B is a schematic diagram of NAD+ on THP-1 cell toxicity determination. DETAILED DESCRIPTION

[0011] The application will be further described in conjunction with the specific embodiments. The embodiments provided below are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The embodiments provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute a limitation on the application in any way.

[0012] The experimental methods in the following examples are not specifically described, and the reagents used in the following examples are commercially available or prepared according to conventional methods in the art, and are laboratory grade. If not specified, the experimental methods and experimental conditions used in the following examples are conventional experimental methods and experimental conditions in the art, which can be referred to relevant experimental manuals, known literature or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the application pertains.

[0013] As mentioned before, at present, there is no specific drug for the infection of monkeypox virus. In view of this, the present application provides small molecule compounds for inhibiting monkeypox virus and applications thereof.

[0014] In one specific embodiment of the present application, the use of Theaflavin-3'-Gallate or NAD+ in the preparation of products for preventing or treating monkeypox virus infection is provided.

[0015] The applicant previously constructed a library of 179 plasmids expressing different proteins of MPXV and carried out screening, and found that the protein OPG188 encoded by the OPG188 gene of MPXV can significantly inhibit the natural immune process of monkeypox virus.

[0016] The OPG188 of MPXV is a protein with a length of 503 amino acids, and the N-terminal contains a Poxin-like protein. Studies have found that Poxin-like proteins exist in the genomes of many viruses and animals, and viruses containing Poxin-like proteins can mostly inhibit the activation of the cGAS-STING antiviral signaling pathway by degrading 2'3'-cGAMP. For example, the Poxin-like protein encoded by the B2R gene of VACV can degrade 2'3'-cGAMP and inhibit the transmission of the cGAS-STING antiviral signal. This suggests that the OPG188 protein of MPXV has great potential to antagonize natural antiviral immunity.

[0017] The applicant uses the FDA and TargetMol databases of the computer big data platform for virtual screening, takes the active site of the Poxin in the OPG188 protein as the center of the docking area of the virtual screening, generates the grid file required for docking, screens out candidate compounds that may interact with the OPG188 protein, and further screens out the compounds Theaflavin-3'-Gallate and NAD+ with the highest affinity to OPG188 through candidate gene experiments, and verifies the effect through cell experiments and molecular experiments.

[0018] In some embodiments of the present application, the product is a drug.

[0019] In some embodiments of the present application, the drug is a single active ingredient drug, and the active ingredient thereof is Theaflavin-3'-Gallate or NAD+.

[0020] In some embodiments of the present application, the drug is a compound drug, and the active ingredient thereof contains Theaflavin-3'-Gallate or NAD+.

[0021] In some embodiments of the present application, the drug further contains at least one other non-drug active ingredient.

[0022] In some embodiments of the present application, the other non-drug active ingredient includes a pharmaceutically acceptable carrier, excipient and / or diluent.

[0023] In some embodiments of the present application, the other non-drug active ingredient includes a pharmaceutically compatible inorganic or organic acid or base, a polymer, a copolymer, a block copolymer, a monosaccharide, a polysaccharide, an ionic and non-ionic surfactant or lipid, and a pharmacologically harmless salt.

[0024] In some embodiments of the present application, the dosage form of the drug includes true solution, colloid, microparticle, emulsion, suspension, tablet, capsule, drop pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder, inclusion compound, landfill, patch and rub.

[0025] In some embodiments of the present application, the concentration of Theaflavin-3'-Gallate in the drug is 90-110 μM, preferably 100 μM.

[0026] In some embodiments of the present application, the concentration of NAD+ in the drug is 90-110 μM, preferably 100 μM.

[0027] In another embodiment of the present application, a pharmaceutical composition for preventing or treating monkeypox virus infection is provided, which comprises Theaflavin-3'-Gallate or NAD+.

[0028] In another embodiment of the present application, a method for inhibiting monkeypox virus infection for non-therapeutic purposes in vitro is provided, which comprises administering an effective dose of the small molecule compound of the first aspect to an infected system or an infected individual, and after a predetermined time, the level of monkeypox virus in the serum of the infected system or the infected individual is significantly reduced or reduced to an undetectable level.

[0029] Theaflavin-3'-Gallate described herein is a red tea theaflavins monomer, the Chinese name is theaflavin-3'-gallate, the chemical formula is C 36 H 28 O 16 , and the CAS number is 28543-07-9.

[0030] NAD+ described herein is a coenzyme that transfers hydrogen ions and participates in redox reactions in cells, the Chinese name is nicotinamide adenine dinucleotide, the chemical formula is: C 21 H 27 N7O 14 P2, and the CAS number is 53-84-9.

[0031] The main reagents in the following examples: Dual Luciferase Reporter Assay Kit was purchased from Promega, item number: DL101-01; Theaflavin-3'-Gallate was purchased from MCE, item number: HY-N0244; NAD+ was purchased from MCE, item number: HY-B0445; TRizol reagent was purchased from Thermo, item number: 15596026CN; 2X Universal SYBR Green Fast qPCR Mix was purchased from Abclonal, item number: RK21203; ABScript II cDNA First-Strand Synthesis Kit was purchased from Abclonal, item number: RK20400; Cell Counting Kit (CCK-8) CCK-8 kit was purchased from YEASEN, item number: 40203ES80.

[0032] The source and construction of plasmids in the following examples: pcDNA3.1 empty vector was purchased from Yobio, item number: VT1010; IFNβ reporter gene plasmid IFN-Beta_pGL3 (hereinafter referred to as pGL3) was purchased from Addgene, item number: 102597; pRL-TK reporter gene plasmid pRL-TK was purchased from Biyun Tian, item number: D2760; cGAS overexpression plasmid pCMV-HA-N-cGAS (human) (referred to as cGAS) was purchased from Solabio, item number: VT011154; STING overexpression plasmid pCMV-STING1 (human)-3xFLAG-Neo (referred to as STING) was purchased from Solabio, item number: VT031984.

[0033] OPG188 overexpression plasmid pCDH-OPG188 (referred to as OPG188): The CDS region of OPG188 (Gene ID: 928917) gene mRNA was synthesized by gene fragment synthesis method, and the CDS region of OPG188 was constructed into the multiple cloning site insertion region of pCDH-CMV-MCS-EF1-Puro vector (referred to as pCDH) by the experimental method of vector construction in molecular biology. The specific method is as follows: pCDH-CMV-MCS-EF1-Puro plasmid (purchased from Youbao Biology, item number: VT1480) was used as the template, pCDH-F (pCDH-F: 5'-GGATCCGCGGCCGCGAAGGATCT-3', SEQ ID NO: 1) and pCDH-R (pCDH-R: 5'-GATTTAAATTCGAATTCGCTAGCTC-3', SEQ ID NO: 2) were used as the upstream and downstream primers, and ApexHF HS DNA polymerase premix-FCL (purchased from Aikewo Biology, product item number: AG12212) was used for amplification of the target fragment. The amplification product was separated by 0.8% agarose gel electrophoresis, and the target band was recovered by gel recovery kit (the agarose gel recovery kit was purchased from Omega, item number: D2500-02) as a linearized vector. The CDS region of OPG188 (Gene ID: 928917) gene mRNA was used as the template, OPG188-F (OPG188-F: 5'-GCTAGCGAATTCGAATTTAAATCATGTTTTACGCACACGCTTTC-3', SEQ ID NO: 3) and OPG188-R (OPG188-R: 5'-GATCGCAGATCCTTCGCGGCCGCTTAAAATTTTATATGTGACA-3', SEQ ID NO: 4) were used as the upstream and downstream primers, and ApexHF HS DNA polymerase premix-FCL (purchased from Aikewo Biology, product item number: AG12212) was used for amplification of the target fragment. The amplification product was separated by 1% agarose gel electrophoresis, and the target band was recovered by gel recovery kit (the agarose gel recovery kit was purchased from Omega, item number: D2500-02) as an insertion fragment. The amplified linearized vector and the insertion fragment were connected by ClonExpress II One Step Cloning Kit seamless cloning kit (purchased from Novozyme, item number: C112-01). The OPG188 overexpression plasmid pCDH-OPG188 was constructed.

[0034] Cells and viruses used in the following examples: HEK293T cells, THP-1 cells were purchased from ATCC cell bank, monkeypox virus (Clade IIb) was preserved in the P3 laboratory of the First Affiliated Hospital of Zhejiang University.

[0035] Culture medium used in the following examples: DMEM medium (Gibco, catalog number: 11965092), RPMI 1640 medium (Gibco, catalog number: 11875119), fetal bovine serum (Gibco, catalog number: A5669701).

[0036] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0037] Example 1 This example provides a method for virtually screening candidate compounds of OPG188 protein using FDA and TargetMol database of computer big data platform, the specific steps are as follows: The crystal structure of OPG188 protein (PDB number: 8P44) was automatically obtained through RCSB Protein Data Bank database (http: / / www.rcsb.org / ). Except for A, B chain, all redundant atoms were deleted, and the protein structure was carefully processed through several steps, including: repairing residues, protonation and adding partial charge under AMBER ff14SB force field. The DMS tool was used to generate the protein surface with a probe radius of 1.4 Å. The co-crystal ligand (WY8) was used to define the binding pocket (i.e., the active site of Poxin in OPG188 protein), and the Sphgen module of UCSF Chimera was called to generate a small ball filled in the site. The center of the box around the small ball was set at (23.328, 44.353, 63.707), and the size was (27.964, 26.556, 28.556). The DOCK 6.9 program was used for semi-flexible docking, 10000 different conformational orientations were generated for each compound, and the Grid scoring was used for binding force evaluation. Cluster analysis (RMSD threshold 2.0 Å) was performed on the candidate conformations, and finally one scored best conformation was output. After manual analysis and selection, 17 compounds for purchase were screened out according to the score, as shown in Table 1.

[0038] Table 1 17 compounds

[0039] Example 2 In the virtual docking screening of Example 1, 17 candidate compounds were obtained. In order to further narrow down the range of target compounds, this embodiment further screened small molecule compounds inhibiting the function of monkey pox virus OPG188 by detecting the expression of reporter gene IFNβ and qPCR experiment.

[0040] (1) IFNβ reporter gene experiment HEK293T cells were cultured to 90% confluence, and then cell transfection was performed. The transfection was divided into three groups. The first group was a negative control pcDNA3.1 group: transfected with pGL3 plasmid + pRL-TK plasmid + pcDNA3.1. The second group was a positive control pCDH group: transfected with pGL3 plasmid + pRL-TK plasmid + pCMV-STING1 (human)-3xFLAG-Neo plasmid + pCMV-HA-N-cGAS (human) plasmid + pCDH-CMV-MCS-EF1-Puro plasmid. The third group was an experimental group: divided into 18 parallel holes, and transfected with pGL3 plasmid + pRL-TK plasmid + pCDH-OPG188 plasmid + pCMV-STING1 (human)-3xFLAG-Neo plasmid + pCMV-HA-N-cGAS (human) plasmid. After 6 h of cell transfection, the first group and the second group were not treated and continued to be cultured for 18 h. In the third group, PBS and 17 kinds of small molecule compounds were added, respectively. The volume of PBS and small molecule compounds added was 10 μL, the volume of culture medium was 1 mL, the final concentration of each compound in the culture medium was 100 μM, and after 18 h of compound incubation and treatment, the activation of IFNβ reporter gene was detected by an enzyme marker.

[0041] The study found that MPXV can escape the clearance of host innate immunity by inhibiting the production of type I interferon. Host innate immunity is the first line of defense against viral infection and control of viral replication in the early stage of viral infection. It mainly activates downstream signaling pathways by recognizing viral nucleic acids (DNA or RNA), initiates type I interferon, inflammatory cytokine expression, and induces cell death, etc., to resist viral infection. Cyclic GMP-AMP synthase (cGAS) is an important DNA receptor in the cytoplasm, which can recognize viral DNA and activate the adaptor protein in the downstream signaling pathway to initiate type I interferon expression and activate antiviral innate immunity. The activation of STING will activate NF-κB, although the activation of STING to NF-κB is lower than that of other inflammatory pathway cascades. After the activation of the cGAS-STING signaling pathway, activated IRF3 and NF-κB enter the nucleus to initiate the expression of type I interferon and inflammatory cytokines, and play an antiviral role. The applicant constructed 179 plasmid libraries expressing different proteins of MPXV and screened them, and found that the protein OPG188 encoded by the OPG188 gene of MPXV can significantly inhibit the activation of the cGAS-STING antiviral signal. Knocking down the expression of OPG188 can promote the activation of the interferon antiviral signaling pathway and the expression of antiviral genes after monkeypox virus (MPXV) infection. Further, it is indicated that OPG188 of monkeypox virus is an important factor for inhibiting the host antiviral immune process during viral infection.

[0042] The IFNβ reporter gene contains the promoter of IFNβ, followed by the firefly luciferase. After the reporter gene is transfected into cells, when the IFNβ signaling pathway in the cells is activated, the transcription factor inducing the expression of IFNβ will bind to the promoter region of IFNβ, thereby starting the expression of downstream firefly luciferase. Therefore, the expression amount of firefly luciferase can reflect the expression of IFNβ in the cells. The TK promoter of the pRL-TK reporter gene is not induced for expression, and the expression amount of sea cucumber luciferase after the stable expression promoter can reflect the transfection efficiency of the IFNβ reporter gene cGAS, STING, etc.

[0043] As shown in Figure 1 , compounds No. 2 and No. 13 can eliminate the inhibitory effect of OPG188 on interferon expression. The two compounds are NAD+ and Theaflavin-3'-Gallate, respectively. The chemical structural formula of Theaflavin-3'-Gallate is shown as A in Figure 2 , and the chemical structural formula of NAD+ is shown as B in Figure 2As shown in B in the diagram. The interaction pattern between OPG188, constructed using molecular docking, and the compound Theaflavin-3'-Gallate and NAD+ is shown in the diagram. Figure 2 As shown in C and D, Theaflavin-3'-Gallate and NAD+ both occupy the catalytic active sites of OPG188.

[0044] To further explore whether there is a direct interaction between NAD+ and Theaflavin-3'-Gallate and OPG188, the expression of type I interferon IFNB1 and the antiviral genes ISG54 / ISG56 was detected by qPCR to further verify whether they inhibit the function of monkeypox virus OPG188. The specific steps are as follows: The pCMV-STING1(human)-3×FLAG-Neo plasmid (STING), pCMV-HA-N-cGAS(human) plasmid (cGAS), pCDH-OPG188 plasmid (OPG188), and the empty vector pCDH-CMV-MCS-EF1-Puro (pCDH) were used as references. Figure 3 The cells were transfected into HEK293T cells as shown in the diagram. Six hours after transfection, the cells were treated with Theaflavin-3'-Gallate and NAD+ at a concentration of 100 μM for each compound. After incubation for 18 hours, the mRNA transcriptional changes of IFNB1, ISG54, and ISG56 were detected by qPCR. The qPCR primer sequences are shown in Table 2.

[0045] Table 2 Primer Sequences

[0046] The results are as follows Figure 3 As shown in A, B, and C, the expression detection of type I interferon IFNB1 and antiviral gene ISG54 / ISG56 revealed that NAD+ and Theaflavin-3'-Gallate could also partially eliminate the inhibitory effect of OPG188 on the expression of type I interferon IFNB1 and antiviral gene ISG54 / ISG56.

[0047] Example 3 Determination of the inhibitory effects of Theaflavin-3'-Gallate and NAD+ on monkeypox virus in THP-1 cells 10 5THP-1 cells were plated in 6-well cell culture plates, and Theaflavin-3'-Gallate and NAD+ were added at a final concentration of 100 μM. The control group was treated with the corresponding dose of normal saline, and then infected with MPXV virus. After 96 hours, the virus in the supernatant was collected, and 1 μL of the supernatant was added to a new 6-well cell culture plate to infect new THP-1. After 12 hours, the cells were collected, and the expression of monkeypox virus OPG187, OPG188, and OPG189 genes in the cells was detected to explore the inhibitory effect of Theaflavin-3'-Gallate and NAD+ on the replication of monkeypox virus. OPG187, OPG188, and OPG189 are all genes of monkeypox virus, and the detection results of multiple genes are consistent, which can better indicate that the inhibitor has an antiviral effect. The detection primers are shown in Table 2. The results are shown in Figs. 1A, 1B, and 1C. Figure 4 As shown in Figs. 1A, 1B, and 1C, Theaflavin-3'-Gallate and NAD+ have obvious inhibitory effects on the replication of monkeypox virus.

[0048] Example 4 This example explores the effect of Theaflavin-3'-Gallate and NAD+ on the viability of THP-1 cells.

[0049] 10 4 THP-1 cells were plated in 96-well cell culture plates, and different concentrations (0 μM, 20 μM, 50 μM, 100 μM, 200 μM, and 400 μM) of Theaflavin-3'-Gallate and NAD+ were added. After 24 hours of culture, 10 μL of CCK8 was added, and the cells were incubated at 37°C for 1 hour. The absorbance at OD450 was detected by an enzyme marker. The results are shown in Figs. 2A and 2B. Figure 5 As shown in Figs. 2A and 2B, Theaflavin-3'-Gallate and NAD+ have no obvious cytotoxicity to THP-1 cells at a treatment concentration of 400 μM or less. Finally, it should be noted that the above description is only for the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements of some parts. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of Theaflavin-3'-Gallate or NAD+ in the preparation of a product for preventing or treating monkeypox virus infection.

2. Use according to claim 1, wherein The product is a medicament.

3. Use according to claim 2, wherein the compound is ###0002### The medicament is a single active ingredient medicament, and the active ingredient thereof is Theaflavin-3'-Gallate or NAD+.

4. The use according to claim 2, wherein the compound is ###0002### The medicament is a compound medicament, and the active ingredient thereof comprises Theaflavin-3'-Gallate or NAD+.

5. Use according to any one of claims 3 to 4, characterized in that, The effective concentration of Theaflavin-3'-Gallate or NAD+ is 90-110 μM.

6. The use according to claim 2, wherein The medicament further comprises at least one other non-pharmacologically active ingredient.

7. Use according to claim 6, wherein The other non-pharmacologically active ingredient includes a pharmaceutically acceptable carrier, excipient and / or diluent.

8. Use according to claim 7, wherein the compound is ###0002### The other non-pharmacologically active ingredient includes a pharmaceutically compatible inorganic or organic acid or base, polymer, copolymer, block copolymer, monosaccharide, polysaccharide, ionic and non-ionic surfactant or lipid, pharmacologically harmless salt.

9. The use according to claim 2, wherein The dosage form of the medicament includes true solution, colloid, microparticle, emulsion, suspension, tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, freeze-dried powder injection, inclusion compound, landfill, patch and liniment.

10. A method of inhibiting monkeypox virus infection in vitro for non-therapeutic purposes, characterized in that, It comprises administering an effective dose of Theaflavin-3'-Gallate or NAD+ to an infected system or an infected individual.

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