Application of Compound SH in the Preparation of Broad-Spectrum Anti-Pox Virus Drugs

Abstract

This invention provides the application of compound SH in the preparation of broad-spectrum anti-orthopox virus drugs, belonging to the field of biomedical technology. This invention selects monkeypox virus (MPXV-B.1-China-C-tan-CQ01), vaccinia virus WR strain (WR), recombinant vaccinia virus Tiantan strain (TTV) expressing green fluorescent protein, and mousepox virus (ECTV) as representative strains. Through studies on the inhibitory activity of viral nucleic acid replication and infectious viral particles, it was found that compound SH has broad-spectrum anti-orthopox virus activity, laying the foundation for the development of broad-spectrum anti-orthopox virus drugs and possessing significant development value and broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of compound SH in the preparation of broad-spectrum anti-acne virus drugs. Background Technology

[0002] Poxviruses are the largest known linear DNA viruses by genome size. They have a wide host range, and most can cause zoonotic diseases, manifesting as localized or systemic purulent skin lesions. Mammalian poxviruses can be divided into eight genera: Orthopoxvirus (OPXV), Goatpoxvirus, Fowlpoxvirus, Parapoxvirus, Swinepoxvirus, Rabbitpoxvirus, Molluscum Poxvirus, and Yatapoxvirus. Among these, Orthopoxvirus is the most numerous, has the widest host range, and is the most damaging genus within the subfamily Mammalia. Variola virus (VARV), Monkeypox virus (MPXV), Cowpox virus (CPXV), and Vaccinia virus (VACV) within this genus are all capable of infecting humans. Smallpox viruses pose a serious threat to humans. Among them, smallpox, caused by smallpox virus, is a highly contagious disease with a mortality rate of over 50%. Monkeypox virus, cowpox virus, and vaccinia virus, which are related to smallpox, can all infect humans across species and have important public health significance.

[0003] Currently, the main anti-poxvirus drugs used clinically are acyclic monophosphate nucleotide analogs cidofovir (CDV) and brincidofovir (HDP-CDV), as well as the broad-spectrum envelope protein inhibitor tecovirimat (ST-246). CDV has adverse effects in clinical use, including low oral bioavailability, requiring intravenous administration, and significant nephrotoxicity. HDP-CDV is a lipophilic prodrug of CDV with oral activity and stronger in vitro and in vivo activity than CDV, but it has adverse effects such as elevated liver transaminases, nausea, and vomiting. ST-246's widespread use is limited by its high cost and the susceptibility to mutations at membrane protein sites. Therefore, there is an urgent need to develop novel and highly effective anti-poxvirus drugs to address poxvirus outbreaks.

[0004] Compound SH, also known as "Siai Te San," is an anti-HIV drug developed by Researcher Luo Shide and others at the Kunming Institute of Botany starting in 1987. It is formulated from five traditional Chinese herbs selected from over 20 herbs exhibiting strong anti-HIV activity: licorice root, artemisia annua, mulberry bark, astragalus root, and safflower, following the principle of "principal, assistant, adjuvant, and guide" in traditional Chinese medicine. In Phase I / II clinical trials, Compound SH reduced viral load by more than 0.5 log in 14-35% of HIV-positive patients. Studies have shown that the combined use of Compound SH and the antiretroviral drug atazanavir (ATV) can enhance the inhibitory effect on HIV-1 protease activity. However, there are currently no reports of Compound SH being used as a drug for preparing anti-monkeypox virus, nor any reports of it being used as a broad-spectrum anti-orthopox virus drug. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of compound SH in the preparation of a broad-spectrum anti-orchid virus drug. Compound SH can significantly inhibit the nucleic acid replication of four orchid viruses, namely MPXV-B.1-China-C-tan-CQ01, WR, TTV and ECTV, in vitro, and can inhibit the production of infectious viral particles.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of compound SH in the preparation of broad-spectrum anti-vaccinia virus drugs.

[0008] Preferably, the orthopoxvirus includes monkeypoxvirus, vaccinia virus WR strain, recombinant vaccinia virus Tiantan strain expressing green fluorescent protein, and mousepoxvirus.

[0009] Preferably, the compound SH inhibits the nucleic acid replication of vaccinia virus.

[0010] Preferably, the compound SH inhibits the production of infectious viral particles.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] This invention selected representative orthopoxvirus strains, including monkeypoxvirus (MPXV-B.1-China-C-tan-CQ01), vaccinia virus WR strain (WR), recombinant vaccinia virus Tiantan strain (TTV) expressing green fluorescent protein, and mousepoxvirus (ECTV). A systematic in vitro pharmacodynamic study of compound SH against orthopoxvirus was conducted, focusing on its inhibitory activity on viral nucleic acid replication and infectious viral particles. The study explored the potential application of compound SH in the preparation of broad-spectrum anti-poxvirus drugs. The results showed that compound SH significantly inhibited the nucleic acid replication of four orthopoxvirus strains (MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV) in vitro and suppressed the production of infectious viral particles, demonstrating potential for broad-spectrum anti-poxvirus pharmacodynamic activity and holding significant importance for the prevention and treatment of poxviruses. Attached Figure Description

[0013] Figure 1 : Results of cytotoxicity assay of compound SH in Vero cells;

[0014] Figure 2 The inhibitory effect of different concentrations of compound SH on the proliferation of recombinant vaccinia virus Tiantan strain TTV virus expressing green fluorescent protein was tested using the fluorescent focus inhibition method.

[0015] Figure 3 The inhibitory effects of different concentrations of compound SH on the replication of MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV nucleic acids in Vero cells were detected by qPCR.

[0016] Figure 4 Plaque assay results of different concentrations of compound SH on the inhibitory effect of MPXV-B.1-China-C-tan-CQ01, WR, TTV and ECTV viral replication in Vero cells;

[0017] Figure 5 The inhibitory effects of different concentrations of compound SH on the replication of MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV viruses in Vero cells were analyzed by plaque assay. Detailed Implementation

[0018] This invention provides the application of compound SH in the preparation of a broad-spectrum anti-orthopox virus drug. The orthopox virus includes monkeypox virus (MPXV-B.1-China-C-tan-CQ01), vaccinia virus WR strain (WR), recombinant vaccinia virus Tiantan strain (TTV) expressing green fluorescent protein, and mousepox virus (ECTV).

[0019] This invention systematically investigated the in vitro pharmacodynamics of compound SH against orthopoxvirus by focusing on its inhibitory activity against viral nucleic acid replication and infectious viral particles, exploring the potential application of compound SH in the preparation of broad-spectrum anti-poxvirus drugs. The results showed that compound SH significantly inhibited the nucleic acid replication of four orthopoxvirus strains (MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV) in vitro and suppressed the production of infectious viral particles, demonstrating potential for broad-spectrum anti-poxvirus pharmacodynamics and holding significant importance for the prevention and treatment of poxviruses.

[0020] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0021] Experimental methods not specified in the following examples are generally performed under conventional conditions, such as those described in commonly used reference books in the field, such as "Molecular Cloning: A Laboratory Manual" (3rd edition, Science Press, 2005), or according to the conditions recommended by the reagent manufacturers.

[0022] In a specific embodiment of the present invention, the selected Siatesan (compound SH) was purchased from Zetai Ltd., Thailand; and the Vero cells were purchased from ATCC.

[0023] Example 1

[0024] This embodiment included a cytotoxicity assay for compound SH: the sensitive cell line used for the orthopoxvirus involved was Vero cells. To test the safe dosage concentration of compound SH, this embodiment used the CCK-8 assay to detect the toxicity of compound SH in sensitive cell lines.

[0025] The detection principle is as follows: CellCountingKit-8 (CCK-8) utilizes the water-soluble tetrazolium salt developed by Dojindo. (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt), The orange-yellow formazan dye produced by the oxidation-reduction reaction of intracellular dehydrogenases is soluble in tissue culture medium, and the amount of formazan produced is directly proportional to the number of viable cells. The number of viable cells is determined by the absorbance value (OD value) at 450 nm; the higher the OD value, the stronger the cell activity and the lower the toxicity of the drug being tested.

[0026] The specific procedures are as follows: One day before the experiment, the cells were incubated at 2×10⁻⁶ cells / day. 4Seed cells in 96-well plates until they reach 90% confluence. Weigh 500 mg of selenophora and add 10 mL of ddH2O. Vortex and sonicate to dissolve to a concentration of 50 mg / mL as the stock solution. Store at 4°C. When using, dilute MEM containing 2% fetal bovine serum to concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL. Add 100 μL of the drug diluent to each well, with three replicates for each concentration. Simultaneously, set up cell controls and blank controls. The cell control wells contain no drug solution, and the blank control wells contain neither cells nor drug solution. Incubate the cells in a 37°C incubator with 5% CO2 for 72 hours. After 72 hours, cell viability was tested according to the CCK-8 reagent kit instructions: CCK-8 reagent was diluted 1:10 with serum-free medium, the supernatant of the 96-well plate was discarded, and 100 μL of reagent dilution was added to each well. The 96-well plate was incubated in an incubator for 90 minutes, and the absorbance at 450 nm was measured using a microplate reader. The test was stopped when the OD value of the cell control well was around 1.5.

[0027] The cell activity inhibition rate was calculated according to formula (1): with the drug concentration of compound SH as the abscissa and the cell proliferation inhibition rate as the ordinate, the mean and standard deviation were calculated using GraphpadPrism9 software to fit the curve. After converting the drug concentration to a logarithmic value, the cytotoxic CC of compound SH was calculated. 50 .

[0028] Cell viability = [(As-Ab) / (As-Ac)] × 100% (1)

[0029] Where As = absorbance of experimental wells (containing cells, culture medium, CCK8, and drugs); Ab = absorbance of control wells (containing cells, culture medium, and CCK8, but excluding drugs); Ac = absorbance of blank wells (containing culture medium and CCK8, but excluding cells and drugs).

[0030] See results Figure 1 The curves show the fitted curves of Vero cell inhibition under different concentrations of compound SH. The horizontal axis represents the drug concentration of compound SH, and the vertical axis represents the cell inhibition rate. The results show that compound SH produces a 50% cytotoxic concentration (CC) in Vero cells. 50 The concentration was 97.18 μg / ml, indicating good safety.

[0031] Example 2

[0032] This embodiment tested the in vitro broad-spectrum anti-vaccinia virus activity of compound SH:

[0033] Preparation of stock solution: Weigh 500mg of Siatesan, add 10ml of ddH2O, vortex and sonicate to dissolve to 50mg / ml as the stock solution, store at -20℃.

[0034] (1) Detection of the inhibitory effect of compound SH on TTV virus by fluorescence focus inhibition method

[0035] Vero cells were divided into 2×10 4 Cells were seeded in 96-well plates and cultured in MEM medium containing 10% fetal bovine serum for 16 hours until the cell density reached 80%. The culture medium was then aspirated and replaced with MEM medium containing 2% fetal bovine serum. To evaluate the anti-poxvirus efficacy of compound SH, 2× volumes of drug solution were prepared. Compound SH working solution was added to the corresponding Vero cell wells at concentrations of 100 μL per well (100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, and 3.125 μg / ml). Simultaneously, control wells (without drug) and virus control wells (with virus only) were set up. Within 1.5 hours after drug addition, Poxvirus was diluted to 100 PFU / 100 μL with 2% fetal bovine serum MEM, and 100 μL was added to each well, i.e., each well contained 100 PFU of virus. The final drug concentrations were 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, and 1.5625 μg / ml, respectively. Cells were cultured at 37°C in a 5% CO2 incubator for 72 h, and then photographed using a fluorescent dot counter. The inhibition results of compound SH on TTV fluorescent focus formation are shown in [Figure number missing]. Figure 2 .

[0036] Depend on Figure 2 It can be seen that as the concentration of compound SH drug increases, the brightness and size of TTV virus fluorescent plaques gradually decrease, indicating that the addition of the drug inhibits the growth of the virus and has a certain dose-effect relationship.

[0037] (2) The inhibitory effect of compound SH on four strains of virus, MPXV-B.1-China-C-tan-CQ01, WR, TTV and ECTV, was detected by real-time quantitative q-PCR.

[0038] Vero cells were divided into 2×10 4Cells were seeded in 96-well plates and cultured in MEM medium containing 10% fetal bovine serum for 16 hours until the cell density reached 80%. The culture medium was then aspirated and replaced with MEM medium containing 2% fetal bovine serum. To evaluate the anti-poxvirus efficacy of compound SH, 2× volumes of drug solution were prepared, and the compound SH working solution was added at concentrations of 100 μL per well: 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, and 3.125 μg / ml. A dose of 1 μL was added to the corresponding Vero cell wells, with corresponding control wells (no drug added) and virus control wells (virus added only). Within 1.5 hours after drug addition, the poxvirus was diluted to 100 PFU / 100 μL with 2% fetal bovine serum MEM, and 100 μL was added to each well, resulting in 100 PFU of virus per well. The final drug concentrations were 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, and 1.5625 μg / ml, respectively. Cells were incubated at 37°C in a 5% CO2 incubator for 72–96 hours.

[0039] The viral replication level was determined by detecting the universal viral target genes of MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV using quantitative real-time q-PCR. Viral DNA was extracted according to the instructions of the Xi'an Tianlong Viral DNA / RNA Nucleic Acid Extraction Kit and detected by q-PCR using Taq Pro U+ Multiple Probe qPCR Mix (QN213). The primer and probe sequences for viral detection are as follows:

[0040] For orthopoxvirus WR, TTV, and ECTV, the universal primer and probe sequence for orthopoxvirus E3L was used for detection. The primer and probe sequence is as follows:

[0041] The upstream primer sequence (E3L-qF) is SEQ ID NO:1:

[0042] 5'-ATCCTCTCTCATTGATTTTTCGCGGGA-3';

[0043] The downstream primer sequence (E3L-qR) is SEQ ID NO:2:

[0044] 5'-TGGAGAAGCGAGAAGTTAATAAAGC-3';

[0045] The probe sequence (q-probe) is 5'-HEX-SEQ ID NO:3-BHQ1-3':

[0046] 5'-HEX-TCGTCGGAGCTGTACACCATAGCAC-BHQ1-3'.

[0047] The reaction mixture consisted of 10 μL of 2×Taq Pro U+Multiple Probe qPCR Mix, 0.4 μL of upstream primer, 0.4 μL of downstream primer, 0.4 μL of probe, and 2 μL of DNA template, which was then brought to a final volume of 20 μL with sterile double-distilled water.

[0048] The reaction parameters were: 37℃ for 2 min, 95℃ for 30 s per cycle; 95℃ for 10 s, 60℃ for 30 s per cycle, for 40 cycles. Each sample was tested in triplicate. The CT values ​​of the samples were then statistically analyzed. The viral copy number in the samples was calculated by substituting the measured CT values ​​into the standard curve. The calculation was performed according to formula (2):

[0049] Viral replication inhibition rate (%) = (Virus control group - Drug control group) / Virus control group × 100% (2)

[0050] For monkeypox virus MPXV-B.1-China-C-tan-CQ01, monkeypox virus F3L-specific primers and probes were used for detection. The primer and probe sequences are as follows:

[0051] The upstream primer sequence (F3L-qF) is SEQ ID NO:4:

[0052] 5'-CTCATTGATTTTTCGCGGGATA-3',

[0053] The downstream primer sequence (F3L-qR) is SEQ ID NO:5:

[0054] 5'-CTCATTGATTTTTCGCGGGATA-3',

[0055] The probe sequence (q-probe) is 5'-FAM-SEQ ID NO:6-MGB-3':

[0056] 5'-FAM-CATCAGAATCTGTAGGCCGT-MGB-3'.

[0057] The reaction mixture consisted of 12.5 μL of 2×Probe qPCR Mix, 1 μL of upstream primer, 1 μL of downstream primer, 0.5 μL of probe, and 5 μL of DNA template, which was then brought to a final volume of 25 μL with sterile double-distilled water.

[0058] The reaction parameters were: 50℃ for 3 min, 95℃ for 10 min for one cycle; 95℃ for 15 s, 60℃ for 60 s for 40 cycles. Each sample was repeated 3 times. Finally, the CT value of the sample was counted, and the viral copy number in the sample was calculated by substituting the measured CT value into the standard curve. The calculation was performed according to formula (2).

[0059] See results Figure 3 , Figure 3 In the figure, A to D represent the inhibitory effects of compound SH on four viral strains: MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV, respectively. The horizontal axis represents the drug concentration of compound SH, and the vertical axis represents the qPCR inhibition rate. The curve fitted by the dark dots in the boxes represents the efficiency of viral replication inhibition. qPCR results showed that compound SH effectively inhibited the replication of MPXV-B.1-China-C-tan-CQ01 in Vero cells, achieving a 50% inhibition rate (EC50) for 100 PFU of MPXV-B.1-China-C-tan-CQ01 replication (EC50). 50 The concentration was 24.62 μg / ml; Compound SH effectively inhibited WR replication in Vero cells, achieving a 50% inhibition rate (EC50) for WR replication at 100 PFU. 50 The concentration was 23.55 μg / ml; Compound SH effectively inhibited TTV replication in Vero cells, achieving a 50% inhibition rate (EC50) for 100 PFU of TTV replication. 50 The concentration was 27.23 μg / ml; Compound SH effectively inhibited ECTV replication in Vero cells, achieving a 50% inhibition rate (EC50) for 100 PFU of ECTV replication. 50 It was 23.68 μg / ml.

[0060] (3) The plaque inhibition method was used to detect the inhibitory effect of compound SH on four types of orthopoxviruses: MPXV-B.1-China-C-tan-CQ01, WR, TTV and ECTV.

[0061] Vero cells were divided into 2×10 5Cells were seeded in 12-well plates and cultured in MEM medium containing 10% fetal bovine serum for 16 hours until the cell density reached 90%. The culture medium was then aspirated and replaced with MEM medium containing 2% fetal bovine serum. To evaluate the anti-orchiopsoriasis virus efficacy of compound SH, 2× volumes of drug solution were prepared. Compound SH working solution was then added to the corresponding Ver at concentrations of 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, and 3.125 μg / ml per well (500 μL per well). In the cell wells, corresponding cell control wells without drugs and virus control wells with only virus were set up simultaneously; within 1.5 h after drug addition, the poxvirus was diluted to 100 PFU / 500 μL with 2% fetal bovine serum MEM, and 500 μL was added to each well, that is, each well contained 100 PFU of virus; at this time, the final drug concentrations were 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, and 1.5625 μg / ml, respectively. The cells were cultured at 37℃ in a 5% CO2 cell incubator for 72 h to 96 h; then 1000 μL of 0.1% crystal violet-4% paraformaldehyde fixative was added to each well, and the cells were fixed and stained for 30 min. The staining-fixative solution was discarded, and 1 ml of ddH2O was added to each well for washing. After drying, the empty spots were photographed and counted. The count was calculated according to formula (3):

[0062] Drug inhibition rate = (number of virus control plaques - number of experimental well plaques) / number of virus positive control plaques × 100% (3)

[0063] See results Figure 4 , Figure 4 In the table, A to D represent the plaque inhibition results of compound SH on four types of orthopoxviruses: MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV, respectively. The results show that as the concentration of compound SH increases, the number of plaques for MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV gradually decreases.

[0064] (4) Fitting curve and calculating EC 50

[0065] Plotting the concentration of compound SH on the x-axis and the viral replication inhibition rate on the y-axis, a curve was constructed using the mean and standard deviation of the inhibition efficiency calculated with Graphp ad Prism 9 software. The EC50 of compound SH was then calculated after converting the drug concentration to logarithm. 50 .

[0066] See results Figure 5 , Figure 5In the figure, A to D represent the inhibitory effects of compound SH on the viral replication of four poxviruses: MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV, respectively. The horizontal axis represents the drug concentration of compound SH, and the vertical axis represents the plaque inhibition rate. The curve fitted by the dark dots in the boxes represents the efficiency of viral replication inhibition. The results showed that compound SH could effectively inhibit the replication of MPXV-B.1-China-C-tan-CQ01 in Vero cells, achieving a 50% inhibition rate of MPXV-B.1-China-C-tan-CQ01 replication at 100 PFU (median effective concentration EC50). 50 The concentration was 23.82 μg / ml; Compound SH effectively inhibited WR replication in Vero cells, achieving a 50% inhibition rate (EC50) for WR replication at 100 PFU. 50 The concentration was 29.98 μg / ml; Compound SH effectively inhibited TTV replication in Vero cells, achieving a 50% inhibition rate (EC50) for 100 PFU of TTV replication. 50 The concentration was 29.70 μg / ml; Compound SH effectively inhibited ECTV replication in Vero cells, achieving a 50% inhibition rate (EC50) for 100 PFU of ECTV replication. 50 The concentration was 47.30 μg / ml.

[0067] (5) Calculation of the index SI

[0068] Drug toxicity CC 50 With EC 50 The ratio of the two values ​​can be used to calculate the Selective Index (SI), which is an important indicator for judging drug efficacy. A Selective Index > 1 indicates that the drug is effective and safe; the higher the SI value, the safer and more effective the drug. Based on the cytotoxicity test results obtained in Example 1, CC... 50 The half-maximal effective concentration (EC50) of the drug for virus inhibition obtained in steps (2) and (4) of Example 2. 50 Further calculate the selection index SI = CC 50 / EC 50 The selectivity index (SI) of SH compound SH against MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV in Vero cells was obtained. The results are shown in Table 1.

[0069] Table 1. In vitro anti-orchidoid virus efficacy of compound SH.

[0070]

[0071] As shown in Table 1 and Figure 3As shown, the selection indices (SI) of SH compound SH in Vero cells, measured by qPCR, for MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV were 3.95, 4.13, 3.57, and 4.1, respectively.

[0072] As shown in Table 1 and Figure 5 As shown, the selection indices (SI) of SH compound SH in Vero cells against MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV, as measured by the plaque method / fluorescent focus inhibition method, were 4.11, 3.24, 3.27, and 2.05, respectively.

[0073] Based on the above results, compound SH has good antiviral effects against four orthopoxvirus strains: MPXV-B.1-China-C-tan-CQ01, WR, TTV, and ECTV.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Siatesan in the preparation of broad-spectrum anti-poxvirus drugs, characterized in that, The orthopoxviruses are monkeypoxvirus, vaccinia virus WR strain, recombinant vaccinia virus Tiantan strain expressing green fluorescent protein, and mousepoxvirus.

2. The application according to claim 1, characterized in that, The aforementioned Siatesan inhibits the nucleic acid replication of vaccinia virus.

3. The application according to claim 1, characterized in that, The aforementioned Siatesan inhibits the production of infectious viral particles.

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