Use of remdesivir in the preparation of a preparation for preventing and / or treating peste des petits ruminants virus

Abstract

The application provides a use of remdesivir in preparation of a preparation for preventing and / or treating peste des petits ruminants virus, and belongs to the technical field of biological medicine. The application finds that the use of remdesivir in a safe concentration range can significantly inhibit the replication of the peste des petits ruminants virus in host cells, obviously reduce the expression level of virus-related genes and proteins, and exhibit good antiviral activity. Meanwhile, the inhibitory effect of remdesivir on the PPRV presents obvious dose dependence, and with the increase of the acting concentration, the antiviral effect is significantly enhanced, which indicates that the effect is stable and has strong controllability. Moreover, remdesivir mainly acts on the virus replication stage, can effectively block the virus replication process, thereby reducing the virus infection level and reducing the generation of daughter virus particles. The application provides a new technical approach for the prevention and treatment of peste des petits ruminants, makes up for the deficiency of lack of effective antiviral drugs in the prior art, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of remdesivir in the preparation of agents for the prevention and / or treatment of peste des petits ruminants virus. Background Technology

[0002] Peste des petits ruminants (PPR), also known as sheep plague or pseudo-roughbite, is an acute, highly contagious viral infectious disease caused by the peste des petits ruminants virus (PPRV). This virus belongs to the genus Morbillivirus of the family Paramyxoviridae. PPR virus is an enveloped, negative-sense, single-stranded RNA virus with a genome length of approximately 16 kb. The intact viral particle has a nearly spherical structure with a diameter of approximately 400–500 nm.

[0003] PPRV primarily infects small ruminants such as goats and sheep. Infection can cause systemic multi-organ systemic lesions, with common clinical symptoms including persistent high fever, conjunctivitis, rhinitis and tracheitis, oral mucosal ulcers, severe diarrhea, and pneumonia. The virus enters the viremia stage approximately 2-3 days after infection and causes death within 5-10 days, with mortality rates exceeding 90% in severe outbreaks. Because its clinical symptoms are similar to those of rinderpest, foot-and-mouth disease, bluetongue, and contagious pleuropneumonia in sheep, diagnosis based solely on clinical symptoms is prone to misdiagnosis. Differential diagnosis usually requires laboratory testing, posing significant challenges to livestock production and disease control.

[0004] Currently, peste des petits ruminants (PPR) has spread widely globally, affecting major livestock-producing countries and regions in Africa, Asia, and Europe, posing a serious threat to the global sheep industry. PPR was first discovered in Ngari Prefecture of Tibet Autonomous Region in my country in 2007, and the epidemic has since spread to multiple provinces. As of 2025, at least 29 provinces, autonomous regions, and municipalities in my country have reported PPR outbreaks, causing significant economic losses to the sheep industry.

[0005] Currently, the prevention and control of peste des petits ruminants (PPR) mainly relies on immunization with attenuated live vaccines. However, vaccine immunization still has certain limitations, such as limited duration of protection, insufficient vaccination coverage in some areas, and difficulty in timely control during outbreaks. Furthermore, in recent years, the PPR virus has undergone continuous genetic evolution, with genomic mutations and recombination occurring in its strains, which to some extent increases the difficulty of vaccine control. At the same time, specific drugs for treating PPR remain scarce, and related treatment methods are relatively limited.

[0006] In animal disease prevention and control systems, small-molecule chemical drugs offer advantages such as ease of use, low cost, and rapid onset of action. They can serve as an important intervention during disease outbreaks, complementing vaccination strategies and thus improving disease control effectiveness. Therefore, screening and obtaining novel antiviral components that can effectively inhibit the replication of peste des petits ruminants (PPR) virus and are used for the prevention and treatment of PPR is of great significance for improving the comprehensive prevention and control system for PPR. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide the use of remdesivir in the preparation of agents for the prevention and / or treatment of peste des petits ruminants (PPRV). The present invention has found that remdesivir can significantly inhibit PPRV replication, thereby effectively preventing and treating PPRV.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of remdesivir in the preparation of agents for the prevention and / or treatment of peste des petits ruminants virus.

[0009] Preferably, the molecular formula of remdesivir is C2. 18 H 27 NO3, the structural formula is: Formula 1.

[0010] Preferably, the effective concentration of remdesivir is 0.1~0.5mM.

[0011] Preferably, remdesivir works by inhibiting the replication of peste des petits ruminants virus.

[0012] Preferably, the dosage form of the preparation includes tablets, capsules, suspensions, solutions, emulsions, injections, ointments, gels, films, pellets, granules, or powders.

[0013] The present invention also provides a medicament for the prevention and / or treatment of peste des petits ruminants virus, said medicament comprising remdesivir and a pharmaceutically acceptable carrier.

[0014] Preferably, the concentration of remdesivir in the drug is 0.1~0.5mM.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The remdesivir used in this invention can significantly inhibit the replication of peste des petits ruminants (PPRV) in host cells within a safe concentration range, and significantly reduce the expression levels of virus-related genes and proteins, exhibiting good antiviral activity. Simultaneously, the inhibitory effect of this component on PPRV shows a clear dose-dependent relationship; its antiviral effect significantly increases with increasing concentration, indicating that its action is stable and highly controllable. Further research shows that this component mainly acts on the viral replication stage, effectively blocking the viral replication process, thereby reducing viral infection levels and decreasing the production of progeny virus particles.

[0016] 2. The antiviral agent provided by this invention can serve as an important supplement to vaccine immunization strategies, rapidly exerting an intervention effect during disease outbreaks or immunization gaps, thus helping to improve the overall prevention and control of peste des petits ruminants (PPR). This invention provides a new technical approach for the prevention and treatment of PPR, overcoming the lack of effective antiviral drugs in existing technologies, and has promising application prospects. Attached Figure Description

[0017] Figure 1 To detect the cytotoxicity of different concentrations of remdesivir on Vero cells; Figure 2 The relative expression of the PPRV-N gene in Vero cells under different treatments; Figure 3 The expression levels of PPRV-N protein in Vero cells under different treatments; Figure 4 For TCID 50 To determine the effect of remdesivir on PPRV infection in Vero cells; Figure 5 The relative expression levels of the PPRV-N gene in Vero cells infected at different stages; Figure 6 The expression level of PPRV-N protein in Vero cells infected at different stages; Figure 7 This shows the proliferation of PPRV in Vero cells during the replication phase. Figure 8 The expression level of PPRV-N protein in Vero cells after remdesivir treatment was detected by indirect immunofluorescence assay, where the scale bar is 100 μm; Figure 9 The relative expression of the PPRV-N gene in Vero cells under different doses of remdesivir treatment; Figure 10 The expression of PPRV-N protein in Vero cells under different doses of remdesivir treatment. Detailed Implementation

[0018] This invention provides the use of remdesivir in the preparation of agents for the prevention and / or treatment of peste des petits ruminants virus.

[0019] In this invention, the molecular formula of remdesivir is C 18 H 27 NO3, the structural formula is: Formula 1.

[0020] In this invention, the effective concentration of remdesivir is 0.1~0.5mM, preferably 0.15~0.3mM, and more preferably 0.25mM.

[0021] In this invention, remdesivir works by inhibiting the replication of peste des petits ruminants virus.

[0022] In this invention, the dosage form of the preparation includes tablets, capsules, suspensions, solutions, emulsions, injections, ointments, gels, films, pellets, granules, or powders.

[0023] The present invention also provides a medicament for the prevention and / or treatment of peste des petits ruminants virus, said medicament comprising remdesivir and a pharmaceutically acceptable carrier.

[0024] In this invention, the concentration of remdesivir in the pharmaceutical preparation is 0.1~0.5mM, preferably 0.15~0.3mM, and more preferably 0.25mM.

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] The specific materials and reagents involved in the embodiments of this invention are as follows: Materials: Vero cells (preserved by China Animal Health and Epidemiology Center); PPRV (75-1 strain, China Animal Health and Epidemiology Center); nitrocellulose membrane (NC, Nanjing Novizan Biotechnology Co., Ltd.).

[0027] Reagents: DMEM medium (Thermo Fisher Scientific (China) Co., Ltd.); fetal bovine serum, CCK8, DPEC water, HiScript QRT SuperMix for qPCR (+gDNA wiper) reverse transcription kit, AceQ qPCR SYBR GreenMaster Mix real-time fluorescence kit (Nanjing Novizan Biotechnology Co., Ltd.); penicillin-streptomycin mixture, Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd.); 0.25% trypsin (Beijing Voge Oriental Technology Co., Ltd.); cell lysis buffer, protease inhibitor, 5× non-denaturing protein loading buffer, PBS cell buffer, AF488-labeled goat anti-mouse IgG (H+L) (Shanghai Beyotime Biotechnology Co., Ltd.); 12% protein precast gel, PPRV-N, mouse anti-GAPDH (Qingdao Kolde Science & Trade Co., Ltd.); horseradish peroxidase (HRP)-labeled goat anti-mouse or goat anti-rabbit secondary antibody (Beijing Dongge Boye Biotechnology Co., Ltd., SA00001-2 or SA00001-1); ultrasensitive ECL chemiluminescence kit (NewSemi Biotechnology Co., Ltd.).

[0028] Example 1: Detection of Vero Cytotoxicity of Remdesivir

[0029] To determine the safe concentration of remdesivir, Vero cells were prepared at a concentration of 1 × 10⁻⁶. 4 Remdesivir was seeded at a density of 100 μL / mL in 96-well plates. Each well was then incubated with 100 μL of DMEM medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The plates were incubated at 37°C in a 5% CO2 incubator until approximately 80% confluence. Different final concentrations (0.1, 0.25, and 0.5 mM) of remdesivir were added to the cells and cultured for 24 h, with 100 μL of DMSO as a negative control. After incubation, the original cell culture medium was removed and replaced with 100 μL of DMEM medium containing 10 μL of CCK-8. After incubation at 37°C for 2 h, the absorbance was measured at 450 nm. Cell viability at different concentrations of remdesivir was calculated, with DMSO treatment considered as 100%. The results are shown below. Figure 1 As shown.

[0030] Depend on Figure 1 It is known that remdesivir at a final concentration of 0.1–0.25 mM has no cytotoxicity to Vero.

[0031] Example 2: Effect of Remdesivir on PPRV Virus Infection

[0032] Vero cells were administered at a rate of 6 × 10⁻⁶. 5Vero cells were seeded at a density of 1 / mL in 6-well plates. 1 mL of DMEM medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin was added to each well (2 mL per well in 6-well plates, 1 mL per well in 12-well plates). The plates were incubated at 37°C with 5% CO2 until the cells reached 70% confluence. The original medium was discarded, and the cells were washed three times with PBS. Vero cells were treated with DMSO, 0.1 mM, or 0.25 mM remdesivir for 2 h, then inoculated with PPRV virus containing the corresponding concentration of remdesivir (MOI = 1). The plates were incubated at 37°C with 5% CO2 for 2 h, then the virus solution was discarded, and the cells were washed three times with PBS to remove any uninfected virus. DMSO, 0.1 mM, or 0.25 mM remdesivir were added to the DMEM cell culture medium, and the treated Vero cells were added. The plates were then incubated at 37°C with 5% CO2 for 24 h.

[0033] Add 500 μL of Buffer CRL to a 6-well cell plate containing discarded cell culture medium, ensuring it fully covers the cell surface. Then, repeatedly pipette the cells to detach them. Transfer all the lysate to RNA Columns I (each sample in a 1.5 mL ep ep tube), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the waste liquid. Add 500 μL of Buffer RWA (containing anhydrous ethanol) to RNA Columns I, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the waste liquid. Add 500 μL of Buffer RW (containing anhydrous ethanol) to RNA Columns I, centrifuge at 12,000 rpm (13,400 × g) for 1 min, and discard the waste liquid. Return RNA Columns I to the collection tube and centrifuge at 12,000 rpm (13,400 × g) for 1 min to prevent ethanol contamination. Carefully transfer RNA Columns I to a new 1.5 mL RNase-free centrifuge tube. Add 20 μL of RNase-free ddH2O to the center of the adsorption column membrane, incubate at room temperature for 1 min, and then centrifuge at 12,000 rpm (13,400 × g) for 1 min to elute RNA. After adjusting the RNA concentration (800 ng / μL), use the HiScript Q RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit to generate complementary DNA (cDNA) as the RT-PCR template. The 20 μL reverse transcription system contains 1000 ng RNA, 4 μL of reverse transcriptase 5×qRT SuperMix, and DEPC water to make up to 20 μL. The reverse program is 37℃ for 30 min. Using cDNA as the RT-PCR template, the Nanjing Novizan AceQ qPCR SYBR Green Master Mix reagent was used, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Viral RNA was normalized using GAPDH mRNA and analyzed by 2... -△△CT The PPRV-N RNA content was determined by relative quantitative RT-PCR. Primers and reaction systems are shown in Tables 1 and 2. A Bio-Rad real-time PCR instrument (Bio-Rad Laboratories, USA) was used. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, for 40 cycles. After amplification, melting curve analysis was performed using the following program: 95℃ for 15 s, 60℃ for 1 min; 95℃ for 15 s. Primer specificity was determined based on the specificity of amplification at 85±0.8℃ Tm. Each sample was tested in triplicate, and the average value was taken.

[0034] Table 1 Primer Sequence Information for Quantitative Real-Time PCR

[0035] Table 2. Real-time PCR reaction system

[0036] Vero cells from 6-well plates treated similarly were collected using Triton X-100 protein lysis buffer containing 1× protease inhibitor. Cell samples were centrifuged at 4°C, 12000 rpm for 15 min to obtain the supernatant protein from the cell lysis buffer. Protein concentration was determined using the BCA method, and the loading volume of the target protein was adjusted accordingly. 5× non-denaturing protein loading buffer was added, and the samples were boiled at 100°C for 10 min followed by brief centrifugation. The loading volume was adjusted using the same concentration of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a standard, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG) was performed. E) Transfer the protein sample to a 0.22 μm nitrocellulose membrane (NC) at a constant voltage of 110V for 30 min; block with TBST buffer containing 5% (v / v) skim milk powder at room temperature for 1 h; incubate with mouse anti-PPRV-N and mouse anti-GAPDH as primary antibodies at room temperature for 1 h; wash three times with TBST for 5 min each time, then add horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody and incubate at room temperature for 1 h; wash three times with TBST, and perform Western blot analysis on the protein levels of PPRV-N and GAPDH using a high-sensitivity ECL chemiluminescence kit. The results are as follows: Figures 2-4 As shown.

[0037] Depend on Figures 2-4 The results show that 0.25 mM remdesivir significantly inhibited the expression of PPRV-N gene and protein. This indicates that 0.25 mM remdesivir significantly inhibits PPRV virus infection.

[0038] Example 3: Effects of Remdesivir on Different Stages of PPRV Virus Infection (Western blot, quantitative RT-PCR, and TCID) 50 )

[0039] Vero cells were divided into 6×10 5Cells were seeded at a density of [number] cells / mL in 6-well plates. 2 mL of DMEM medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin was added to each well. The plates were incubated at 37°C with 5% CO2 until cells reached 80% confluence. The medium was discarded, and the cells were washed three times with PBS to remove residual serum. To determine the effect of remdesivir on different stages of PPRV infection, remdesivir was added during the adsorption, internalization, and replication stages of PPRV infection.

[0040] Adsorption group: 100 μL of DMSO and 0.25 mM remdesivir were added to Vero cells and PPRV (MOI=1) were inoculated for virus infection. The cells were placed in a 4°C refrigerator to allow the virus to adsorb without entering the cells. After 1 h, the cells were washed three times with PBS to remove the unadsorbed virus. 2 mL of DMEM cell culture maintenance medium containing 2% serum was added and the cells were incubated in a 37°C, 5% CO2 incubator for 24 h.

[0041] Internalization group: Vero cells were seeded with PPRV (MOI=1), and 100 μL of DMSO and 0.25 mM remdesivir were added to the cells respectively. The cells were incubated at 37°C. After 1 h, the cells were washed three times with PBS to remove remdesivir and unadsorbed virus. 2 mL of DMEM maintenance medium containing 2% serum was added, and the cells were incubated at 37°C and 5% CO2 for 24 h.

[0042] Replication group: Vero cells were seeded with PPRV (MOI=1), incubated at 37°C and 5% CO2 for 2 hours, washed three times with PBS to remove unadsorbed virus, and 2 mL of DMEM cell culture medium containing 2% serum was added. 100 μL of DMSO and 0.25 mM remdesivir were added to the cells, and the cells were incubated at 37°C and 5% CO2 for 24 hours.

[0043] Vero cells were digested and diluted to an appropriate density with 10% FBS DMEM medium, and then cultured at a density of 2 × 10⁶ cells / mL. 4 Cells were seeded at a density of 103 / mL in 96-well plates and incubated at 37°C with 5% CO2 until they reached 80% confluence. Twelve 1.5mL EP tubes were prepared, and 900µL of DMEM medium containing 2% serum was added to each tube. The pre-dissolved virus solution was vortexed to mix. 100µL of PPRV was then extracted and added to the first EP tube containing 900µL of DMEM medium with 2% serum. After vortexing, 100µL of the mixture was added to the next tube, and so on. The last tube did not contain PPRV but was supplemented with 100µL of DMEM medium containing 2% serum as a negative control. The virus dilution range was 10-1. -1 ~10 -11Discard the culture medium from the 96-well plates, wash the cells three times with PBS, and then inoculate each dilution of virus solution into columns 1-12 of the 96-well cell culture plates, with 8 replicates per dilution, and 100 µL per well. Incubate the 96-well plates at 37°C in a 5% CO2 incubator, observing cell morphology twice daily and marking wells showing cytopathic effects. Continue observation for 5 days, then record the number of wells with cytopathic effects (CPE) for each dilution, and calculate the TCID of the virus using the Reed-Muench method. 50 The result is as follows Figures 5-7 As shown.

[0044] Depend on Figures 5-7 It was found that, compared with the DMSO control group, remdesivir mainly exerted its effect during the viral replication phase of infection. A final concentration of 0.25 mM remdesivir significantly inhibited PPRV replication.

[0045] Example 4: Indirect immunofluorescence assay to verify the effect of remdesivir on PPRV replication

[0046] Vero cells were divided into 6×10 5 Vero cells were seeded at a density of 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in each well of a 6-well plate. The plates were incubated at 37°C and 5% CO2 until the cells reached 80% confluence. The culture medium was discarded, and the cells were washed three times with PBS to remove residual serum. Vero cells were seeded with PPRV (MOI=1) and incubated at 37°C and 5% CO2 for 2 hours. After washing three times with PBS to remove unadsorbed virus, 2 mL of DMEM cell culture medium containing 2% serum was added. 100 μL of DMSO and 0.25 mM remdesivir were added to the cells, and the plates were incubated at 37°C and 5% CO2 for 24 hours. Cells were then washed three times with PBS, fixed with 4% paraformaldehyde for 15 min each time, washed three times with PBS, and then 1 mL of diluted murine monoclonal antibody against PPRV N protein (1:1000) was added and incubated at 37°C for 1 h. Cells were then washed three times with PBS, and 1 mL of diluted FITC-labeled goat anti-mouse secondary antibody (1:5000) was added under light-protected conditions and incubated at 37°C for 45 min. Cells were then washed three times with PBS, and 1 mL of freshly prepared DAPI dilution was added to each well, and nuclei were stained for 15 min under light-protected conditions at room temperature. Cells were then washed three times with PBS and observed using an inverted fluorescence microscope. The results are shown below. Figure 8 As shown.

[0047] Depend on Figure 8It can be seen that, compared with the DMSO control group, the number of fluorescence in the remdesivir experimental group was significantly less than that in the control group, indicating that 0.25 mM remdesivir significantly inhibited PPRV replication.

[0048] Example 5: Remdesivir within the safe concentration range can dose-dependently inhibit PPRV replication.

[0049] Vero cells were divided into 6×10 5 Vero cells were seeded at a density of 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in each well of a 6-well plate. The plates were incubated at 37°C with 5% CO2 until the cells reached 80% confluence. The culture medium was discarded, and the cells were washed three times with PBS to remove residual serum. Vero cells were seeded with PPRV (MOI=1) and incubated at 37°C with 5% CO2 for 2 hours. After washing three times with PBS to remove unadsorbed virus, 2 mL of DMEM cell culture medium containing 2% serum was added. Then, 100 μL of DMSO and different final concentrations of remdesivir (0.15 mM, 0.2 mM, and 0.25 mM) were added to the cells, and the plates were incubated at 37°C with 5% CO2 for 24 hours. RNA and total protein were then extracted from the cells according to the method in Example 2. The transcriptional level of viral genes and the expression level of proteins were detected by RT-qPCR and Western blot, respectively. The results are shown below. Figure 9 and Figure 10 As shown.

[0050] Depend on Figure 9 and Figure 10 It can be seen that, compared with the DMSO treatment group, remdesivir at different final concentrations can significantly inhibit PPRV replication, and the inhibitory effect is more significant with the increase of remdesivir concentration.

[0051] This invention has discovered that remdesivir can inhibit the replication of PPRV in host cells, thereby effectively preventing and treating infection with peste des petits ruminants virus.

[0052] 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. Application of Remdesivir in the preparation of agents for the prevention and / or treatment of peste des petits ruminants virus.

2. The application according to claim 1, characterized in that, The molecular formula of remdesivir is C 18 H 27 NO3, the structural formula is: Formula 1.

3. The application according to claim 1, characterized in that, The effective concentration of remdesivir is 0.1~0.5mM.

4. The application according to claim 1, characterized in that, Remdesivir works by inhibiting the replication of peste des petits ruminants virus.

5. The application according to claim 1, characterized in that, The dosage forms of the preparations include tablets, capsules, suspensions, solutions, emulsions, injections, ointments, gels, films, pellets, granules, or powders.

6. A medicament for the prevention and / or treatment of peste des petits ruminants virus, characterized in that, The drug comprises remdesivir and a pharmaceutically acceptable carrier.

7. The pharmaceutical preparation according to claim 6, characterized in that, The concentration of remdesivir in the drug is 0.1~0.5mM.

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