Application of galidesivir in inhibiting rabies virus in vitro

Abstract

The present invention relates to the field of pharmaceutical technology and specifically discloses the use of galidesivir for inhibiting rabies virus in vitro. The present invention finds that galidesivir can effectively inhibit the proliferation of RABV in cells while exhibiting minimal cytotoxicity. Galidesivir can be used as a remedy for rabies wounds, maximally eliminating the virus from wounds, and thus provides a new approach to the treatment of rabies virus.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of galidesivir in inhibiting rabies virus in vitro. Background Art

[0002] Rabies is a zoonotic infectious disease caused by the rabies virus (RABV). Symptoms of infection include fear of wind and water, pharyngeal muscle spasms, and progressive paralysis. As a highly preventable but incurable infectious disease, it poses a significant threat to human health and safety. With the continuous innovation and advancement of antiviral drugs, many viral diseases such as hepatitis B, hepatitis C, and AIDS have been effectively controlled, and even drug treatment for Ebola has shown positive results. Currently, there are no effective therapies or antiviral drugs for rabies. Rabies vaccines, immunoglobulins, and the Milwaukee protocol cannot completely block viral infection. Therefore, finding effective and rational medications to treat rabies has become a hot topic in rabies prevention and treatment.

[0003] Galidesivir (BCX4430) is an adenosine analog and direct-acting antiviral drug that disrupts viral RNA-dependent RNA polymerase (RdRp) activity. Galidesivir has in vitro activity against a variety of RNA viral pathogens, including filoviruses and emerging infectious agents such as MERS-CoV, SARS-CoV, and SARS-CoV-2. In vivo, galidesivir has demonstrated activity against a variety of experimental infections following intramuscular, intraperitoneal, and oral administration. In nonclinical studies, galidesivir has demonstrated some efficacy against Ebola, Marburg, Rift Valley fever, and yellow fever virus infections, but there are currently no reports of its efficacy against rabies virus. Summary of the Invention

[0004] One of the objects of the present invention is to provide a new method for resisting rabies virus.

[0005] The present invention has found that galidesivir can effectively inhibit RABV in vitro and has a long effective duration of action, thereby proposing the present invention.

[0006] Specifically, the present invention demonstrated that galidesivir has weak cytotoxicity through cell experiments. Even when 250 μM of galidesivir was added to the cell culture medium, CCK8 assays showed no significant decrease in cell viability. The present invention also found that galidesivir significantly inhibited rabies virus proliferation in multiple cell lines, such as BHK-21 cells (a clone of hamster kidney cells) and N2a cells (mouse neuroblastoma cells), indicating that the drug's effects are broad, rather than cell-specific.

[0007] The present invention further tested the effective duration of galidesivir after the virus enters the cells. The virus titer in the cell supernatant was detected 24, 48 and 72 hours after the virus entered the cells. It was found that as the time after infection continued, the drug still had an effective ability to clear the virus. When tested at 72 hours, the drug still had a significant inhibitory effect on RABV.

[0008] At the same time, the present invention tested the inhibitory effect of galidesivir on multiple RABV strains, including the laboratory-fixed virulent strain CVS-11 and the rabies virus street strain SC16, which was isolated from the brain of a dog that died of rabies in 2016, passaged in mouse brains, adapted to N2a cell growth, and obtained a high titer. The experimental results showed that 250 μM galidesivir could reduce the titers of both viruses, indicating that galidesivir can effectively inhibit multiple RABV strains.

[0009] Given that favipiravir and ribavirin have excellent in vitro RABV inhibition effects, the present invention also compared the inhibitory effects of galidesivir with these agents. By adding certain concentrations of favipiravir, ribavirin, and galidesivir to cells after virus infection, the results showed that galidesivir significantly inhibited RABV replication and was more effective than favipiravir, with a higher SI index than ribavirin. Galidesivir also had better inhibitory efficacy and safety than remdesivir.

[0010] Specifically, the technical solutions of the present invention are as follows:

[0011] In a first aspect, the present invention provides a use of galidesivir in the preparation of an inhibitor, which can inhibit the proliferation of rabies virus in vitro.

[0012] In the application of the present invention, when the inhibitor is used, the effective concentration of galidesivir is 50-250 μmol / L, preferably 250 μmol / L.

[0013] The inhibitor of the present invention can achieve the effect of inhibiting rabies virus in various cells, and has a long action time and ideal effect.

[0014] In a second aspect, the present invention provides a method for inhibiting rabies virus in vitro for purposes other than disease diagnosis and treatment, which uses galidesivir to inhibit rabies virus.

[0015] In the method of the present invention, the concentration of galidesivir used is 50-250 μmol / L, preferably 250 μmol / L.

[0016] The method of the present invention can be used for treating rabies wounds to prevent the rabies virus from harming the human body as much as possible, and can also be used for non-disease diagnosis and treatment purposes such as the development of rabies virus drugs.

[0017] The beneficial effects of the present invention are at least:

[0018] The present invention discovered that galidesivir can effectively inhibit the proliferation of RABV in cells with minimal cytotoxicity. Therefore, it can be used as a drug for rabies wound treatment, maximizing the elimination of the virus at the wound site. Currently, drugs for treating rabies virus are still in the exploratory stage. The galidesivir studied in this invention can effectively inhibit the replication of rabies virus in vitro at a dose of 250 μM, providing new ideas for the treatment of rabies virus.

[0019] Based on the above findings of galidesivir, those skilled in the art can further optimize its molecular structure, optimize its dosage method and time, reduce the dosage, etc., to lay the foundation for the ultimate development of a drug that can treat rabies. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the cytotoxicity test results after different concentrations of galidesivir acted on N2a cell line and BHK-21 cell line for different periods of time.

[0021] Figure 2 Figure 1 shows the results of galidesivir's antiviral activity in BHK-21 cells after CVS-11 infection at different times. Figure A shows the fluorescence image of galidesivir's antiviral activity 24 hours after infection with CVS-11. Figure B shows the statistical graph of galidesivir's antiviral activity 24 hours after infection with CVS-11. Figure C shows the fluorescence image of galidesivir's antiviral activity 48 hours after infection with CVS-11. Figure D shows the statistical graph of galidesivir's antiviral activity 48 hours after infection with CVS-11. Figure E shows the fluorescence image of galidesivir's antiviral activity 72 hours after infection with CVS-11. Figure F shows the statistical graph of galidesivir's antiviral activity 72 hours after infection with CVS-11. Scale bars represent 200 μm.

[0022] Figure 3The results of galidesivir's antiviral activity in BHK-21 cells after SC16 infection at different times. Figures A and B show the fluorescence image of galidesivir's antiviral activity 24 hours after SC16 infection. Figure B shows the statistical graph of galidesivir's antiviral activity 24 hours after SC16 infection. Figure C shows the fluorescence image of galidesivir's antiviral activity 48 hours after SC16 infection. Figure D shows the statistical graph of galidesivir's antiviral activity 48 hours after SC16 infection. Figure E shows the fluorescence image of galidesivir's antiviral activity 72 hours after SC16 infection. Figure F shows the statistical graph of galidesivir's antiviral activity 72 hours after SC16 infection. Scale bars represent 200 μm.

[0023] Figure 4 The results of galidesivir's antiviral activity in the N2a cell line after CVS-11 infection at different times are shown. Figure A shows the fluorescence image of galidesivir's antiviral activity 24 hours after infection with CVS-11. Figure B shows the statistical graph of galidesivir's antiviral activity 24 hours after infection with CVS-11. Figure C shows the fluorescence image of galidesivir's antiviral activity 48 hours after infection with CVS-11. Figure D shows the statistical graph of galidesivir's antiviral activity 48 hours after infection with CVS-11. Figure E shows the fluorescence image of galidesivir's antiviral activity 72 hours after infection with CVS-11. Figure F shows the statistical graph of galidesivir's antiviral activity 72 hours after infection with CVS-11. The scale bar in the figures represents 200 μm.

[0024] Figure 5 The results of galidesivir's antiviral activity in the N2a cell line after SC16 infection at different times are shown. Figure A shows the fluorescence image of galidesivir's antiviral activity 24 hours after SC16 infection in the N2a cell line; Figure B shows the statistical graph of galidesivir's antiviral activity 24 hours after SC16 infection in the N2a cell line; Figure C shows the fluorescence image of galidesivir's antiviral activity 48 hours after SC16 infection in the N2a cell line; Figure D shows the statistical graph of galidesivir's antiviral activity 48 hours after SC16 infection in the N2a cell line; Figure E shows the fluorescence image of galidesivir's antiviral activity 72 hours after SC16 infection in the N2a cell line; and Figure F shows the statistical graph of galidesivir's antiviral activity 72 hours after SC16 infection in the N2a cell line. The scale bar in the figures represents 200 μm.

[0025] Figure 6The results of 48-hour cytotoxicity tests on BHK-21 cell lines treated with ribavirin and favipiravir are shown in the left figure, while the right figure shows the effect of different concentrations of ribavirin on BHK-21 cell viability.

[0026] Figure 7 The results of 48-hour cytotoxicity tests on the N2a cell line after ribavirin and favipiravir treatment are shown in the figure on the left, and the effect of different concentrations of ribavirin on the survival rate of N2a cells is shown on the right.

[0027] Figure 8 Comparison of the rabies virus inhibitory effects of galidesivir, favipiravir, and ribavirin. Figure A shows a fluorescence image of the rabies virus inhibitory effects of galidesivir, favipiravir, and ribavirin, while Figure B shows a bar graph of the viral inhibition rates of galidesivir, favipiravir, and ribavirin. The scale bar represents 200 μm.

[0028] Figure 9 The left figure shows the effect of different concentrations of sofosbuvir on the cell viability of N2a cells and BHK-21 cells, while the right figure shows the effect of different concentrations of sofosbuvir on the cell viability of BHK-21 cells.

[0029] Figure 10 The left figure shows the effect of different concentrations of tenofovir on the cell viability of N2a cells and BHK-21 cells, while the right figure shows the effect of different concentrations of tenofovir on the cell viability of BHK-21 cells.

[0030] Figure 11 The left figure shows the effect of different concentrations of sofosbuvir on viral adsorption in N2a and BHK-21 cells after CVS-11 infection. The right figure shows the effect of different concentrations of sofosbuvir on viral adsorption in BHK-21 cells.

[0031] Figure 12 The left figure shows the effect of different concentrations of tenofovir on viral adsorption in N2a and BHK-21 cells after CVS-11 infection. The right figure shows the effect of different concentrations of tenofovir on viral adsorption in BHK-21 cells.

[0032] Figure 13The results show the effect of different concentrations of sofosbuvir on viral replication in vitro in N2a and BHK-21 cells after CVS-11 infection. The left figure shows the effect of different concentrations of sofosbuvir on viral replication in vitro in N2a cells, and the right figure shows the effect of different concentrations of sofosbuvir on viral replication in vitro in BHK-21 cells.

[0033] Figure 14 The results show the effect of different concentrations of tenofovir on viral replication in vitro in N2a and BHK-21 cells after CVS-11 infection. The left figure shows the effect of different concentrations of tenofovir on viral replication in vitro in N2a cells, and the right figure shows the effect of different concentrations of tenofovir on viral replication in vitro in BHK-21 cells.

[0034] Figure 15 The left figure shows the direct viral killing effect of different concentrations of sofosbuvir on N2a and BHK-21 cells after CVS-11 infection. The right figure shows the direct viral killing effect of different concentrations of sofosbuvir on N2a cells, and the right figure shows the direct viral killing effect of different concentrations of sofosbuvir on BHK-21 cells.

[0035] Figure 16 The results show the direct viral killing effect of different concentrations of tenofovir on N2a and BHK-21 cells after CVS-11 infection. The left figure shows the direct viral killing effect of different concentrations of tenofovir on N2a cells, and the right figure shows the direct viral killing effect of different concentrations of tenofovir on BHK-21 cells. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0037] Unless otherwise specified, the instruments and equipment used in the following examples are all conventional instruments and equipment. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[0038] Example 1

[0039] 1 Materials and Methods

[0040] 1.1 Materials

[0041] 1.1.1 Cells and viruses

[0042] Mouse neuroblastoma N2a cells and hamster kidney cell clone BHK-21 were maintained in our laboratory. Cell nutrient medium: Prepared with Dulbecco's MEM or Gibco BRL, containing 100 mL / L calf serum and 10 mL / L double-stranded antibody. Cell maintenance medium: Prepared with Dulbecco's MEM or Gibco BRL, containing 20 mL / L calf serum. CVS-11 is a classic rabies virus, a fixed strain of the CVS strain adapted to BHK-21 cells. It has strong neurotoxicity and is used as a standard challenge strain in the rapid fluorescent focus inhibition test. It is maintained in this laboratory. The street rabies virus strain SC16 was isolated from the brain of a dog that died of rabies in 2016. After passage in mouse brain and adaptation to N2a cell growth, it has achieved a high titer and is maintained in this laboratory. Information about this strain was published in the November 2016 Journal of Virology, Volume 32, Issue 6, in an article titled "Preparation and Preliminary Application of a Virus Library for Detecting Neutralizing Activity of Rabies Monoclonal Antibodies."

[0043] 1.1.2 Main reagents and instruments

[0044] Galidesivir was purchased from MCE, batch number HY-104077. Galidesivir stock solution: Galidesivir was prepared into a 10 mM stock solution with sterile water and stored at −80°C until use. DMEM high-glucose culture medium, trypsin, double-stranded antibodies, and fetal bovine serum were purchased from GIBCO; anti-rabies virus nucleoprotein fluorescent antibody was purchased from Fujirebio Diagnostics, USA; GoTaq probe-1-Step RT-qPCR mix was purchased from Promega, and the cell proliferation detection kit (CCK8) was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. The inverted microscope was an Olympus CXX4I model from Japan; the fluorescence microscope was an Olympus IX51 model from Japan; the real-time PCR instrument was an ABIQuantStudio 5; the microplate reader was a FLUOstar Omega415-2683; and the cell counter was purchased from ORFLO Technologies, USA.

[0045] 1.2 Methods

[0046] 1.2.1 Determination of drug toxicity on cell viability

[0047] First, the cells were counted and the cell suspension was inoculated. After the cells adhered, galidesivir was added to N2a and BHK-21 cells at final concentrations of 0 μmol / L (DMSO control group), 50 μmol / L, 100 μmol / L, 150 μmol / L, 200 μmol / L, and 250 μmol / L for treatment for 24 h, 48 h, and 72 h. Three repeated experimental groups were set up. After the treatment, 10% of the total volume of CCK-8 reagent was added and incubated in an incubator for 1 h. The absorbance at a wavelength of 450 nm was measured using a microplate reader, which was the OD value. The cell survival rate was calculated using the OD value to express the effect of the drug on cell viability. The formula was: [(experimental well - blank well) / (control well - blank well)] × 100%.

[0048] 1.2.2 Virus titer determination

[0049] Prepare two 96-well plates: one test plate and one dilution plate. Add 200 μL of DMEM to two rows of the dilution plate, then add 50 μL of the viral stock solution. Dilute the cells 5-fold and mix 7-10 times. Transfer 100 μL of the virus stock solution from the dilution plate to the corresponding wells of the test plate. After digestion, add 100 μL of cells to each well. Infect at 37°C, 5% CO₂ for 48 hours. Discard the cell supernatant, and fix the infected cells with 80% cold acetone. Fluorescent foci are counted using direct immunofluorescence. Antigen-positive areas are observed and recorded under a fluorescence microscope. Calculate the viral titer using the following formula: Viral titer = (average of the fluorophore counts in the last four wells with fluorophore development × dilution factor × 1000) / volume of viral supernatant added to each well (FFU / ml).

[0050] 1.2.3 Inhibitory effect of galidesivir on rabies virus replication

[0051] To explore the inhibitory effect of galidesivir on rabies virus, N2a cells and BHK-21 cells were cultured at a rate of 5×10 5 N2a cells and BHK-21 cells were inoculated at a density of 1 / well in a 12-well cell culture plate. CVS-11 was used at an MOI of 1. After 1 hour of infection, the supernatant was discarded and the cells were washed three times with DMEM. Cells were treated with galidesivir at final concentrations of 50 μmol / L, 100 μmol / L, 150 μmol / L, 200 μmol / L, and 250 μmol / L, respectively. Fluorescence foci were read using direct immunofluorescence to determine viral titers. Each group was subjected to three replicates. A DMSO control group was also established, infected with the virus alone.

[0052] In order to explore the inhibitory effect of galidesivir on different rabies viruses, the same experiment was performed on the street strain SC16. The direct immunofluorescence method was used to read the fluorescent focus, and the virus titer determination method was used to determine the virus titer in the cell supernatant to determine the inhibitory effect of galidesivir on different rabies viruses. The experiment was repeated 3 times in each group.

[0053] 2 Results

[0054] 2.1 Toxic effects of galidesivir on N2a and BHK-21 cells

[0055] The experimental results obtained by the cell viability calculation formula of the CCK8 experiment are shown in Figure 1 The results showed that when N2a and BHK-21 cells were treated with galidesivir at a final concentration of less than 250 μmol / L for 24 h, 48 h, and 72 h, no obvious cytotoxic effect was observed on the cells. Therefore, the safe concentration selected in this experiment was less than 250 μmol / L.

[0056] 2.2 Virus titer determination

[0057] According to the calculation formula, the titer of CVS-11 in BHK-21 cell line infection is 10 5.21 FFU / mL, the titer of SC16 is 10 5.06 The CVS-11 titer in N2a cell line infection was 10 FFU / mL. 5.86 FFU / mL, the titer of SC16 is 10 5.43 FFU / mL. In this experiment, the MOI value of CVS-11 and SC16 infection of the two cell lines was established to be 1.

[0058] 2.3 Galidesivir inhibits rabies virus replication in vitro

[0059] After 24h, 48h and 72h of infection of BHK-21 cells with CVS-11 and SC16 strains, different concentrations (50μmol / L, 100μmol / L, 150μmol / L, 200μmol / L, 250μmol / L) of galidesivir were added for treatment. The results showed that different concentrations of galidesivir could effectively inhibit the replication of rabies virus in BHK-21 cells, and the inhibitory effect of 250μmol / L was the most obvious (see Figure 2 and Figure 3At different infection times, the inhibition rates of the 250 μmol / L galidesivir group compared with the CVS-11 infection group were 86.56% (24 h, P < 0.01), 87.57% (48 h, P < 0.01), and 87.61% (72 h, P < 0.01), respectively; and compared with the SC16 infection group, the inhibition rates were 84.67% (24 h, P < 0.01), 84.78% (48 h, P < 0.01), and 85.21% (72 h, P < 0.01), respectively.

[0060] After 24h, 48h and 72h of infection of N2a cells with CVS-11 and SC16 strains, different concentrations (50μmol / L, 100μmol / L, 150μmol / L, 200μmol / L, 250μmol / L) of galidesivir were added for treatment. The results showed that different concentrations of galidesivir could effectively inhibit the replication of rabies virus in N2a cells, and the inhibitory effect of 250μmol / L was the most obvious (see Figure 4 and Figure 5 At different infection times, the inhibition rates of the 250 μmol / L galidesivir group compared with the CVS-11 alone infection group were 88.62% (24h, P<0.01), 90.25% (48h, P<0.01), and 90.66% (72h, P<0.01); compared with the SC16 infection group, the inhibition rates were 85.42% (24h, P<0.01), 86.74% (48h, P<0.01), and 87.25% (72h, P<0.01). The above results indicate that 250 μmol / L galidesivir can effectively inhibit the replication level of RABV, and the inhibitory effect of galidesivir is still significant 72 hours after virus infection.

[0061] Comparative Experiment 1

[0062] The materials and methods involved in this comparative experiment are the same as those in Example 1 unless otherwise specified.

[0063] This comparative experiment first tested the cytotoxicity of ribavirin and favipiravir according to the records in Section 1.2.1 of Example 1 (wherein, the treatment time was set to 48 hours according to the literature). According to the literature, the optimal concentration of favipiravir is 1000 μM and the optimal concentration of ribavirin is 50 μM. Based on this, the treatment concentrations of these two drugs were designed in this comparative experiment, as follows:

[0064] Ribavirin: 0 μmol / L (DMSO control group), 5 μmol / L, 10 μmol / L, 15 μmol / L, 25 μmol / L, 50 μmol / L.

[0065] Favipiravir (T-705): 0 μmol / L (DMSO control group), 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, 1000 μmol / L.

[0066] The specific cytotoxicity results of ribavirin and favipiravir on BHK-21 cell lines are shown in Figure 6 The cytotoxicity results of ribavirin and favipiravir on N2a cell line are shown in Figure 7 .

[0067] In this comparative experiment, N2a cells were further cultured at a rate of 5×10 5 N2a cells were seeded at a density of 1 / well in a 12-well cell culture plate and adsorbed with CVS-11 at an MOI of 1. After 1 hour of infection, the supernatant was discarded and the cells were washed three times with DMEM. Cells were treated with galidesivir, 50 μM ribavirin (optimal concentration published in the literature), or 1000 μM favipiravir (optimal concentration published in the literature) at a final concentration of 250 μmol / L for 48 hours. Fluorescence foci were read using direct immunofluorescence to compare the inhibitory effects of the three agents. A DMSO control group infected with the virus alone was also included. The experiment was repeated three times for each group.

[0068] See the results Figure 8 It can be seen that compared with the CVS-11 virus infection group (DMSO control group), after the N2a cells were treated with 250μmol / L of galidesivir, the virus titer in the galidesivir (BCX4430) treatment group was significantly reduced, with an inhibition rate of 90.04% (**P < 0.01). Compared with the positive control group of Favipiravir (T-705) with an optimal concentration of 1000μM published in other literature, the fluorescence intensity of CVS-11 virus particles in N2a cells treated with galidesivir was also significantly reduced. The results showed that the fluorescence intensity of CVS-11 virus particles in N2a cells treated with galidesivir was not significantly different from that in the ribavirin group (P>0.05), indicating that 250 μmol / L galidesivir can effectively inhibit the replication of CVS-11, and the inhibitory effect is better than that of favipiravir and similar to that of ribavirin.

[0069] The prior art also discloses that the nucleoside analog Remdesivir also has an in vitro inhibitory effect on rabies virus. Therefore, the present invention conducted a safety and efficacy comparison experiment on the four drugs Remdesivir, T705, Ribavirin, and Galidesivir after acting on N2a cells for 48 hours. The specific experimental method is: the cytotoxicity of the drug on cell viability is detected by CCK-8 test to calculate the cytotoxic concentration CC50: cytotoxic concentration, that is, the minimum concentration of the drug required to kill 50% of the host cells; the direct immunofluorescence method is used to read the fluorescent focus and measure the viral titer to calculate the minimum inhibitory concentration IC50 required for the drug to inhibit the pathogen: minimum inhibitory concentration, that is, the concentration of the drug required to inhibit 50% of the pathogen. Finally, the selectivity index SI is calculated: CC50 divided by IC50. The higher the SI index, the more effective the drug is in inhibiting the virus and the less harm it causes to the host cells, so the drug effect is better.

[0070] The research results show that the SI index of remdesivir in inhibiting RABV on N2a cells is 10.36, the SI index of T705 in inhibiting RABV is 8.74, the SI index of ribavirin in inhibiting RABV is 5.68, and the SI index of galidesivir in inhibiting RABV is 50.32 (see Table 1 for specific results), indicating that the inhibitory effect and safety of galidesivir are better than the other three drugs.

[0071] Table 1

[0072]

[0073]

[0074] Comparative Experiment 2

[0075] The materials and methods involved in this comparative experiment are the same as those in Example 1 unless otherwise specified.

[0076] This comparative experiment explored the in vitro inhibitory effects of various nucleoside inhibitors on rabies virus, as follows:

[0077] Sofosbuvir, a nucleoside inhibitor, has demonstrated promising therapeutic effects in the treatment of hepatitis C virus. Tenofovir, another nucleoside inhibitor, is currently used primarily for the antiviral treatment of HIV and HBV. Studies have shown that tenofovir can inhibit the RNA-dependent RNA polymerase of the novel coronavirus, thereby achieving therapeutic effects against the novel coronavirus. Therefore, the present invention also explores the in vitro inhibitory effects of the nucleoside inhibitors sofosbuvir and tenofovir on rabies virus.

[0078] 1. Cytotoxic effects of sofosbuvir and tenofovir on BHK-21 and N2a cells

[0079] Sofosbuvir or tenofovir at final concentrations of 0 μmol / L (DMSO control group), 100 μmol / L, 200 μmol / L, 500 μmol / L, 1000 μmol / L, and 2000 μmol / L were added to BHK-21 and N2a cells and the cells were treated for 48 hours. The absorbance at a wavelength of 450 nm was measured using a microplate reader according to the instructions of the CCK8 cytotoxicity assay kit, which was the OD value. The cell survival rate was calculated using the OD value to indicate the effect of the drug on cell viability.

[0080] The results, calculated using the cell viability calculation formula for the CCK8 assay, showed that sofosbuvir and tenofovir exhibited no significant cytotoxic effects at concentrations of 100 μmol / L, 200 μmol / L, and 500 μmol / L. However, exposure to 1000 μmol / L and 2000 μmol / L resulted in poor cell refractive index, rounding, and detachment, indicating significant cytotoxicity. The cytotoxic effects of the drugs on cells decreased with increasing drug concentrations above 500 μmol / L. Therefore, the safe action concentration selected for this study was 500 μmol / L.

[0081] For detailed results, see Figure 9 and Figure 10 .from Figure 9 It can be seen that the effect of 1000μmol / L sofosbuvir on the survival rate of N2a cells or BHK-21 cells was significantly different from that of 0μmol / L sofosbuvir, *P<0.05 indicates a significant difference; the effect of 2000μmol / L sofosbuvir on the survival rate of N2a cells was extremely significant compared with that of 0μmol / L sofosbuvir, **P<0.01 indicates an extremely significant difference.

[0082] from Figure 10 It can be seen that the effect of 1000μmol / L tenofovir on the survival rate of N2a cells or BHK-21 cells was significantly different from that of 0μmol / L tenofovir, *P<0.05 indicates a significant difference; the effect of 2000μmol / L tenofovir on the survival rate of N2a cells was extremely significant compared with that of 0μmol / L tenofovir, **P<0.01 indicates an extremely significant difference.

[0083] 2. Blocking effect of sofosbuvir and tenofovir on rabies virus adsorption

[0084] N2a cells and BHK-21 cells were cultured at a rate of 5×10 5N2a and BHK-21 cells were seeded at a density of 50 μmol / L (v / well) in a 12-well cell culture plate. Sofosbuvir or tenofovir was added at final concentrations of 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, or 1000 μmol / L, respectively, and preincubated for 2 hours. CVS-11 was added at an MOI of 1 and allowed to adsorb to the cells for 1 hour. Unadsorbed virus and drug were removed, and culture medium was added. The cells were incubated at 37°C for 48 hours. Fluorescent foci were measured using direct immunofluorescence to determine the concentrations of sofosbuvir and tenofovir that blocked viral adsorption. A DMSO control group infected with the virus alone was also included. The experiment was repeated three times for each group.

[0085] After pre-treatment of N2a or BHK-21 cells with sofosbuvir or tenofovir at different concentrations of 50μmol / L, 100μmol / L, 200μmol / L, 500μmol / L, and 1000μmol / L, it was found that sofosbuvir or tenofovir at concentrations below 1000μmol / L could not block the adsorption of rabies virus on N2a or BHK-21 cells.

[0086] For detailed results, see Figure 11 and Figure 12 .from Figure 11 It can be seen that there is no significant difference in the effects of 500μmol / L and 1000μmol / L sofosbuvir on CVS-11 virus-infected N2a and BHK-21 cells compared with the DMSO group, and P>0.05 indicates no statistical difference.

[0087] from Figure 12 It can be seen that there is no significant difference in the effect of 500μmol / L and 1000μmol / L tenofovir on CVS-11 virus infection of N2a and BHK-21 cells compared with the DMSO group, and P>0.05 indicates no statistical difference.

[0088] 3. Inhibitory effects of sofosbuvir and tenofovir on rabies virus replication

[0089] N2a cells and BHK-21 cells were cultured at a rate of 5×10 5 N2a cells and BHK-21 cells were seeded at a density of 1 / well in a 12-well cell culture plate. CVS-11 was adsorbed at an MOI of 1 for 1 hour, the supernatant was discarded, and the cells were washed three times with DMEM. Sofosbuvir or tenofovir was added at final concentrations of 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, or 1000 μmol / L, respectively, and the cells were treated for 48 hours. Direct immunofluorescence was used to measure fluorescent foci and determine the concentrations of sofosbuvir and tenofovir that inhibited viral replication. A DMSO control group infected with the virus alone was also established. The experiment was repeated three times for each group.

[0090] Compared with the CVS-11 virus infection group, after treating N2a or BHK-21 cells with different concentrations of sofosbuvir or tenofovir at 50μmol / L, 100μmol / L, 200μmol / L, 500μmol / L, and 1000μmol / L, it was found that sofosbuvir or tenofovir at concentrations below 1000μmol / L could not inhibit the replication of rabies virus in N2a or BHK-21 cells.

[0091] For detailed results, see Figure 13 and Figure 14 .from Figure 13 It can be seen that there is no significant difference in the effects of 500μmol / L and 1000μmol / L sofosbuvir on CVS-11 virus-infected N2a and BHK-21 cells compared with the DMSO group, and P>0.05 indicates no statistical difference.

[0092] from Figure 14 It can be seen that there is no significant difference in the effect of 500μmol / L and 1000μmol / L tenofovir on CVS-11 virus infection of N2a and BHK-21 cells compared with the DMSO group, and P>0.05 indicates no statistical difference.

[0093] 4. Sofosbuvir and tenofovir directly kill viruses

[0094] N2a cells and BHK-21 cells were cultured at a rate of 5×10 5 / well density inoculated into 12-well cell culture plates, 500 μmol / L sofosbuvir or tenofovir was mixed with MOI=1 of CVS-11 strain and incubated for 1 hour, the drug and virus mixture was used to treat N2a and BHK-21 cells for 1 hour, the unadsorbed virus and drug were removed, culture medium was added, and after incubation at 37℃ for 48 hours, the fluorescence focus was read by direct immunofluorescence method to determine the concentration of sofosbuvir and tenofovir that directly kill rabies virus.

[0095] Compared with the CVS-11 infection group alone, after treating N2a or BHK-21 cells with different concentrations of sofosbuvir or tenofovir of 50μmol / L, 100μmol / L, 200μmol / L, 500μmol / L, and 1000μmol / L, it was found that sofosbuvir or tenofovir at concentrations below 1000μmol / L had no direct killing effect on rabies virus in N2a or BHK-21 cells.

[0096] For detailed results, see Figure 15 and Figure 16 .from Figure 15It can be seen that there was no significant difference in the effects of 50μmol / L, 100μmol / L, 200μmol / L, 500μmol / L and 1000μmol / L sofosbuvir compared with the DMSO group on CVS-11 virus-infected N2a and BHK-21 cells, and P>0.05 indicated that there was no statistical difference.

[0097] from Figure 16 It can be seen that there is no significant difference in the effects of 50μmol / L, 100μmol / L, 200μmol / L, 500μmol / L and 1000μmol / L tenofovir compared with the DMSO group on CVS-11 virus-infected N2a and BHK-21 cells, P>0.05 indicates no statistical difference.

[0098] In summary, galidesivir can effectively inhibit RABV replication in vitro, with 250 μmol / L of galidesivir having the best inhibitory effect. Its inhibitory effect is better than that of favipiravir and similar to that of ribavirin.

[0099] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Use of galidesivir as the sole active ingredient in the preparation of an inhibitor that can inhibit the proliferation of rabies virus in vitro.

2. The use according to claim 1, characterized in that When the inhibitor is used, the effective concentration of galidesivir is 50-250 μmol / L.

3. The use according to claim 2, characterized in that When the inhibitor is applied, the effective concentration of galidesivir is 250 μmol / L.

4. A method for inhibiting rabies virus in vitro for non-disease diagnosis and treatment purposes, characterized in that: Rabies virus suppression is achieved using galidesivir as the sole active ingredient.

5. The method according to claim 4, characterized in that The concentration of galidesivir used is 50-250 μmol / L.

6. The method according to claim 5, characterized in that Galidesivir was used at a concentration of 250 μmol / L.

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