Use of remdesivir in the preparation of a medicine for treating bombyx mori infected with bmcpv

Abstract

The application of Remdesivir in the treatment of BmCPV infected silkworm belongs to the field of molecular biology and virology, when Remdesivir is used with a final concentration of 0.125 mg / mL to treat 1*10^6 BmCPV virus infected silkworm, the expression of BmCPV virus S1 gene can be effectively inhibited, thereby the replication of BmCPV virus is inhibited, and treatment effect is achieved. The present application can provide a theoretical basis for the research and development of silkworm midgut type pyosis treatment drugs, and provide ideas and directions for the research of RNA virus treatment drugs.

Description

Technical Field

[0001] This invention relates to novel applications of remdesivir, particularly its application in the preparation of a treatment for BmCPV-infected silkworms and the drug itself, belonging to the fields of molecular biology and virology. Background Technology

[0002] Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) can infect the midgut cells of silkworms, causing midgut septicemia and resulting in low silkworm yields. It poses a serious threat to sericulture production, and current prevention and control measures for this disease are still limited to disinfection.

[0003] Silkworm cytoplasmic polyhedrovirus (SPCV) is an RNA virus composed of 10 independent dsRNA segments (S1–S10), encoding 6 structural (capsid) proteins (VP1, VP2, VP3, VP4, VP6, VP7) and 3 non-structural proteins. It infects the midgut cells of silkworms, causing midgut septicemia. Midgut septicemia, commonly known as "dry white belly," is primarily transmitted through ingestion. Symptoms include: silkworms consuming mulberry leaves, lethargy, often lying still on the silkworm bed, slow growth and development, significant population disparities, and in later stages, diseased silkworms exhibiting a translucent, "empty head" appearance at the thorax, accompanied by shrinkage, diarrhea, and in severe cases, excrement containing milky white mucus. Disease course: Generally, infected silkworms in their first instar... Onset at age 2; infection at age 2 or older Onset at age 3; infection at age 3 or older The disease manifests in the 3rd and 4th instars; those infected in the 4th instar develop symptoms by the 5th instar; those infected in the mid-to-late 5th instar can generally spin cocoons, but some produce dead cocoons. The disease is characterized by its slow progression and long course, thus classifying it as a chronic infectious disease. In production, infection mostly occurs in the 3rd and 4th instars, gradually increasing in numbers after the 5th instars are fed. The lesions are primarily in the midgut; tearing the back of the silkworm's body wall reveals milky-white folds in the midgut, and in severe cases, the entire midgut appears milky-white.

[0004] After BmCPV virus invades cylindrical cells, the pathological changes in the cells cannot be observed in the early stages of proliferation and replication. However, as the virus continues to replicate and cycle, a series of pathological changes in the infected cylindrical tissue cells can be clearly observed, such as: shedding of the peritrophic membrane, cell deformation, nuclear position shift, vacuolation of cytoplasm, cytoplasm filled with polyhedra, cell collapse, and shedding.

[0005] Remdesivir is a prodrug and has no antiviral activity itself. Currently, there are no reports of remdesivir treating BmCPV-infected silkworms. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide the application of remdesivir in the preparation of a treatment drug for BmCPV-infected silkworms; another purpose of this invention is to provide a drug for treating midgut septicemia in silkworms.

[0007] Technical solution: This invention provides the application of remdesivir in the preparation of a treatment drug for BmCPV-infected silkworms.

[0008] After entering the silkworm's body, remdesivir is taken up by target cells and hydrolyzed by intracellular enzymes. The cleavage of phosphoramide and phosphoester bonds releases the remdesivir monophosphate (RMP) fragment. RMP undergoes continuous phosphorylation in vivo, metabolizing to produce remdesivir triphosphate (RTP). Because RTP is structurally similar to adenine nucleoside triphosphate (ATP), it is mistakenly recognized by newly expressed viral RdRp and incorporated into newly synthesized viral RNA, thus blocking its replication process and achieving the therapeutic goal.

[0009] This invention provides the application of remdesivir as the sole pharmacologically active substance in the preparation of a treatment for BmCPV-infected silkworms.

[0010] Furthermore, remdesivir is selected from any one of the following: monophosphate, sodium salt, toluenesulfonate, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide.

[0011] Furthermore, the BmCPV-infected silkworm is the intestinal septicemic silkworm.

[0012] Furthermore, remdesivir effectively inhibits the expression of the BmCPV S1 gene.

[0013] On the other hand, the present invention provides a drug for treating midgut septicemia in silkworms, the drug comprising remdesivir or its pharmaceutical salt, and a pharmaceutically acceptable carrier.

[0014] Furthermore, the medicinal salt is selected from any one of the following: monophosphate, sodium salt, toluenesulfonate, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of remdesivir.

[0015] Further, the drug comprises remdesivir and DMSO for dissolving remdesivir, wherein the concentration of remdesivir is 0.125–25 mg / mL. Preferably, the concentration of remdesivir is 0.125 mg / mL.

[0016] When remdesivir was used to treat silkworms infected with 1×10^6 BmCPV virus at a final concentration of 0.125 mg / mL, it effectively inhibited the expression of the BmCPV virus S1 gene.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention provides a new theoretical basis and research direction for antiviral treatment of silkworms, and also provides a new strategy for the treatment of midgut septicemia (BmCPV) in silkworms. Attached Figure Description

[0018] Figure 1 The graph shows the relative expression levels of the S1 gene 48 hours after administration of different drug concentrations.

[0019] Figure 2 Image of detoxification enzyme gene detection for drug administration to uninfected individuals;

[0020] Figure 3 Image showing gene detection of detoxification enzymes in patients infected with the virus but not treated with medication;

[0021] Figure 4 Image of detoxification enzyme gene detection for drug administration to infected virus;

[0022] Figure 5 The following is a comparison chart of the fifth instar and sixth day silkworms in each experimental group: 1: Virus-infected drug treatment group; 2: Non-virus-infected drug treatment group; 3: Virus-infected group; 4: Blank control group. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] Example 1

[0025] This invention provides an application of Remdesivir in the treatment of BmCPV-infected silkworms.

[0026] The concentrations of Remdesivir used were 0.125 mg / mL, 0.25 mg / mL, 2.5 mg / mL, 25 mg / mL, and 50 mg / mL.

[0027] This invention provides a drug for treating BmCPV infection in silkworms, using DMSO as the solvent at a concentration of 0.125 mg / mL.

[0028] The technical solution of this invention lies in effectively inhibiting the expression of BmCPV viral genes and hindering viral proliferation, achieved through the following steps:

[0029] (1) Silkworm hatching and breeding

[0030] The silkworm variety selected is the Jingsong × Haoyue hybrid. The silkworm eggs are incubated in an incubation box at 25℃ and 85% relative humidity. After the silkworm eggs turn green, they are treated with light and then collected. After that, they are raised on mulberry leaves normally.

[0031] (2) Infection with BmCPV virus and addition of antiviral drugs

[0032] Silkworms were divided into groups for rearing after reaching the fourth instar. Purified BmCPV virus particles were diluted to 1×10^6 / mL. Mulberry leaves were cut into identical cubes and soaked in the virus solution, while the control group was soaked in water. After air-drying, each group was given the same amount of leaves. Twelve hours after infection, silkworms were fed mulberry leaves containing antiviral drugs (the drugs were dissolved and diluted in DMSO to 0.125 mg / mL, 0.25 mg / mL, 2.5 mg / mL, 25 mg / mL, and 50 mg / mL, then evenly applied to the surface of the mulberry leaves and allowed to dry). Sand was removed before feeding the medicated mulberry leaves. (Antiviral drugs were administered during the fourth instar period.)

[0033] (3) Dissection of midgut tissue

[0034] Midgut tissue was harvested 48 hours after drug administration.

[0035] (4) RNA extraction from tissue and quantitative PCR analysis

[0036] Total RNA was extracted from the midgut and reverse transcribed into cDNA. Gene primers were designed based on the coding region sequences of BmCPV virus and silkworm detoxification enzyme genes in GenBank, and the relative gene expression levels were detected by quantitative PCR.

[0037] (5) Macroeconomic data collection and processing

[0038] During the silkworm rearing process, the weight of silkworms before and after drug administration (i.e., the weight of silkworms in the fourth instar and the weight of silkworms in the fifth instar), the cocoon layer ratio of each group, the mortality rate, and the size of the cocoons were recorded.

[0039] The experimental results are as follows:

[0040] (1) Relative expression level of BmCPV virus S1 gene

[0041] Figure 1 The relative expression levels of the S1 gene 48 hours after administration of different concentrations of the drug were presented. As shown in the figure, the relative expression level of the S1 gene decreased significantly when the final concentration of remdesivir was between 0.125 mg / mL and 25 mg / mL, with the most significant decrease observed at a final concentration of 0.125 mg / mL.

[0042] (2) Detection of detoxification enzyme genes in silkworms

[0043] Figure 2 The patient presented a gene test result for the detoxification enzyme after being administered the drug without being infected with the virus. Figure 3 Genetic testing of the detoxification enzyme in patients infected with the virus but not treated with medication was presented. Figure 4 The results showed the gene detection of the detoxification enzyme after administration of the drug to the infected person. Figure 4 The dosage was 2.5 mg / mL. As shown in the figure, BmCPV infection of silkworms, posing a threat, activates the body's immune system. Remdesivir, however, has extremely low toxicity to silkworms, thus only evoking a weak immune response. Administration of remdesivir after BmCPV infection effectively inhibits viral replication, thereby reducing the harm to silkworms and thus lowering their immune response.

[0044] (3) The therapeutic effects of drugs on each experimental group

[0045] Table 1. The therapeutic effect of the drug of the present invention (0.25 mg / mL) on BmCPV virus.

[0046]

[0047] Note: Blank group – not infected with the virus and not given any medication; Control group – infected with the virus but not given any medication; Experimental group – both infected with the virus and given medication; Mortality rate – number of deaths before the end of age (total number – number of midguts removed) × 100%

[0048] As shown in the table above, BmCPV infection of silkworms has a negative impact on physiological indicators such as silkworm weight and cocoon layer ratio. After administration of the drug, the mortality rate of silkworms was significantly reduced, and all physiological indicators returned to normal. This indicates that the drug of the present invention has a certain therapeutic effect on silkworm diseases caused by BmCPV infection.

[0049] Figure 5 The comparison of the fifth instar and sixth day silkworms in each experimental group was presented. As can be seen from the figure, after the silkworms infected with BmCPV were given the drug, their growth was indeed slightly affected compared with the two control groups (2,4), but it was significantly better than the diseased silkworms that were not given the drug.

Claims

1. Application of Remdesivir as the sole pharmacologically active substance in the preparation of a treatment for BmCPV-infected silkworms.

2. The application according to claim 1, characterized in that, Remdesivir is selected from any one of the following: monophosphate, sodium salt, toluenesulfonate, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide.

3. The application according to claim 1, characterized in that, The drug includes remdesivir and DMSO for dissolving remdesivir, wherein the concentration of remdesivir is 0.125~25 mg / mL.

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