Application of 2'-fucosylated lactose in the preparation of Coxsackie virus inhibitors

Abstract

This invention discloses the application of 2'-fucosyllactose in the preparation of Coxsackievirus inhibitors, belonging to the field of medical preparations. This invention provides the application of 2'-fucosyllactose or its derivatives or pharmaceutically acceptable salts, or substances with 2'-fucosyllactose or its derivatives or pharmaceutically acceptable salts as active ingredients, in any of the following: inhibiting Coxsackievirus, treating diseases caused by Coxsackievirus infection, and improving symptoms caused by Coxsackievirus infection. Based on a Coxsackievirus drug screening model, this invention screened 2'-fucosyllactose, an active ingredient with anti-Coxsackievirus properties, from breast milk components. This invention has significant clinical application value for the prevention of Coxsackievirus and the treatment of diseases following Coxsackievirus infection.

Description

Technical Field

[0001] This invention belongs to the field of medical preparations, specifically relating to the application of 2'-fucosylated lactose in the preparation of Coxsackie virus inhibitors. Background Technology

[0002] Coxsackievirus belongs to the human enterovirus group B (HEV-B). HEV-B presents with a wide range of acute clinical manifestations and is considered a major cause of aseptic meningitis. Furthermore, there is evidence that the pathogenesis of childhood diabetes may also be related to HEV-B, particularly CV-A9, CV-B1, CV-B3, and CV-B5. Coxsackieviruses can be divided into two groups, A (CV-A) and B (CV-B). Coxsackievirus A9 (CV-A9) is the only HEV-B virus in group A (CV-A) and is considered one of the important pathogens of viral encephalitis.

[0003] Although enteroviruses contain many important human pathogens, there are currently no approved drugs for the treatment or prevention of enterovirus infections. Every stage of the viral life cycle could be a potential drug target, but due to concerns about efficacy and safety, very few drugs have entered the global market. Furthermore, vaccines against enteroviruses are not yet widely available, and the diverse range of enteroviruses and their numerous mutant variants present significant challenges for future vaccine design. Given the mutability of small RNA viruses, designing vaccines against all enteroviruses is extremely difficult. Currently, only vaccines targeting poliovirus (PV) and enterovirus A71 (EV-A71) are available in clinical use. Therefore, research into effective antiviral drugs against enteroviruses is of great significance.

[0004] There are currently no reports of 2'-fucosylated lactose inhibiting Coxsackievirus. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to inhibit Coxsackievirus.

[0006] To address the aforementioned technical problems, in a first aspect, the present invention provides the use of 2'-fucosylated lactose or a pharmaceutically acceptable salt thereof, or a composition containing any one of them, said use being (a) and / or (b) and / or (c):

[0007] (a) The use of 2'-fucosylated lactose or a pharmaceutically acceptable salt thereof or a composition containing any one thereof in the preparation of a medicament for the prevention of disease caused by Coxsackievirus or Coxsackievirus infection;

[0008] (b) The use of 2'fucosylated lactose or a pharmaceutically acceptable salt thereof or a composition containing any one thereof in the preparation of a medicament for treating diseases caused by Coxsackievirus or Coxsackievirus infection;

[0009] (c) Use of 2'fucosylated lactose or a pharmaceutically acceptable salt thereof or a composition containing any one thereof in the preparation of Coxsackie virus inhibitors.

[0010] The inhibition of Coxsackievirus can be achieved at the organismal or cellular level.

[0011] The inhibition of Coxsackievirus can be described as inhibiting Coxsackievirus adsorption and entry into host cells.

[0012] Diseases caused by Coxsackievirus infection may include herpetic pharyngitis, acute hemorrhagic conjunctivitis, hand-foot-mouth disease, pleuralgia, myocarditis, pericarditis, and hepatitis. Symptoms caused by Coxsackievirus infection may include fever, sneezing, cough, and rash.

[0013] The inhibition of Coxsackievirus occurs before the virus enters the host or host cells (i.e., before cellular entry). Therefore, 2'-fucosylated lactose is suitable for preparing products for the prevention of diseases caused by Coxsackievirus infection.

[0014] Furthermore, in the above applications, the Coxsackievirus is Coxsackievirus Group A.

[0015] Furthermore, in the above applications, the Coxsackievirus A group refers to Coxsackievirus A9.

[0016] Furthermore, in the above applications, the amino acid sequence of the protein encoded by the complete genome of the Coxsackievirus A9 is SEQ ID No. 2.

[0017] Furthermore, in the above applications, the complete genome nucleotide sequence of the Coxsackievirus A9 is SEQ ID No. 1.

[0018] In one embodiment of the present invention, the Coxsackievirus A9 type is Coxsackievirus A9 strain BUCT01, which has the accession number CGMCC No.20091 at the China General Microbiological Culture Collection Center.

[0019] Furthermore, in the above applications, the drug or Coxsackie virus inhibitor prevents and / or treats diseases caused by Coxsackie virus or Coxsackie virus infection by inhibiting Coxsackie virus adsorption to host cells.

[0020] Furthermore, in the above applications, the drug or Coxsackie virus inhibitor can prevent and / or treat diseases caused by Coxsackie virus or Coxsackie virus infection by inhibiting the entry of Coxsackie virus into host cells.

[0021] Furthermore, in the above applications, the drug or Coxsackie virus inhibitor inhibits the proliferation of Coxsackie virus.

[0022] To address the aforementioned technical problems, in a second aspect, the present invention provides a Coxsackie virus inhibitor comprising the aforementioned 2'-fucosylated lactose or a pharmaceutically acceptable salt thereof, or a composition containing any one of them.

[0023] The structural formula of the 2'-fucosylated lactose is as follows: Figure 10 As shown.

[0024] Furthermore, the aforementioned Coxsackievirus inhibitors also include carrier materials.

[0025] The carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to formulate various dosage forms, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfonate, methylcellulose, and ethylcellulose. For preparing unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. For preparing unit-dose dosage forms into injectable formulations such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added.In addition, colorants, preservatives, flavorings, tasters, sweeteners or other materials may be added to pharmaceutical preparations if necessary.

[0026] The above dosage forms can be used for injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection; respiratory administration, such as through the nasal cavity; and mucosal administration.

[0027] In this invention, the 2'-fucosylated lactose is a human milk oligosaccharide, which is the most abundant oligosaccharide in breast milk.

[0028] This invention utilizes a Coxsackievirus drug screening model to identify 2'-fucosylated lactose, an active drug against Coxsackievirus, from antiviral drugs. This invention has significant clinical application value for the treatment and prevention of Coxsackievirus infection.

[0029] Preservation Instructions

[0030] Bacterial species name: Coxsackievirus;

[0031] Latin name: Coxsackievirus;

[0032] Strain number: CV-A9 isolate BUCT01;

[0033] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0034] The abbreviation for the depository institution is CGMCC.

[0035] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0036] Date of deposit: October 28, 2020;

[0037] Registered with the China National Collection Center (CGMCC) No. 20091; Attached Figure Description

[0038] Figure 1 The relative mRNA expression levels of Coxsackievirus CV-A9 with a multiplicity of infection (MCI) of 0.001 in RD cells were shown after the addition of different concentrations of various breast milk components.

[0039] Figure 2 The final concentrations of 2'-fucosyl lactose (i.e., 10 mg / ml or 5 mg / ml) are shown. Figure 10 The compound shown inhibits viral replication of Coxsackievirus CV-A9 with a multiplicity of infection of 0.001.

[0040] Figure 3 It shows 2'-fucosyl lactose (i.e. Figure 10EC of the compound shown 50 CC 50 And SI.

[0041] Figure 4 It shows 2'-fucosyl lactose (i.e. Figure 10 The results of the dosing time experiment for the compound shown.

[0042] Figure 5 It shows 10 mg / ml of 2'-fucosyl lactose (i.e. Figure 10 The compound shown exhibits an inhibitory effect on the adsorption and uncoating of Coxsackievirus CV-A9, which is infected with a multiplicity of 5.

[0043] Figure 6 It shows 10 mg / ml of 2'-fucosyl lactose (i.e. Figure 10 The compound shown exhibits its inhibitory effect on Coxsackievirus CV-A9 with a multiplicity of 5 at different time points.

[0044] Figure 7 The morphology of RD cells without virus culture for 48 hours and without drug administration is shown.

[0045] Figure 8 The morphology of RD cells without medication is shown 48 hours after infection with the CV-A9 strain BUCT01 with a multiplicity of infection of 0.001.

[0046] Figure 9 The diagram shows the addition of 2'-fucosyl lactose (i.e., 10 mg / ml) to a final concentration of 10 mg / ml. Figure 10 Morphological images of RD cells 48 hours after infection with the compound shown and the CV-A9 strain BUCT01 with an infection multiplicity of 0.001.

[0047] Figure 10 The structural formula of 2'-fucosylated lactose is shown. Detailed Implementation

[0048] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0050] 2'-Fucose-based lactose has the CAS number 41263-94-9 and is a product of Glycome. The structural formula of 2'-fucosylated lactose is as follows: Figure 10 As shown.

[0051] Coxsackievirus A9 strain BUCT01 (also known as CV-A9 strain BUCT01): Isolated and cultured from the feces of children with hand-foot-mouth disease, it was identified by high-throughput sequencing as a Coxsackievirus A9 strain. Whole-genome sequence analysis showed that it shared the highest nucleotide homology (91.38%) with a Coxsackievirus strain isolated in China in 2013 (GenBank accession number: KP289434), and the highest amino acid homology (97.91%) with a Coxsackievirus A9 strain isolated in China in 2013 (GenBank accession number: KP290111). The whole-genome nucleotide sequence of CV-A9 strain BUCT01 is shown in SEQ ID No. 1 (GenBank accession No. MW192795.1, Update Date 24-JAN-2022), and the amino acid sequence encoded by the whole genome is shown in SEQ ID No. 2. The CV-A9 strain BUCT01 was deposited on October 28, 2020, at the China General Microbiological Culture Collection Center (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 20091; the biological material used is BUCT01; the classification name is Coxsackievirus, and its Latin name is Coxsackievirus.

[0052] The following examples used SPSS 19.0 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, with * indicating a significant difference (P < 0.05), ** indicating a highly significant difference (P < 0.01), and *** indicating a highly significant difference (P < 0.001).

[0053] Example 1: Cell Culture and Virus Culture

[0054] Human rhabdomyosarcoma cell line (RD cells) was obtained from the American Type Culture Collection (ATCC, CCL-136) and cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen) at 37°C and 5% CO2.

[0055] The Coxsackievirus A9 strain BUCT01 was propagated in RD cells, and viral titers were determined using plaque assays on RD cells (ATCC CCL-136). All infection experiments were performed in a biosafety level 2 (BLS-2) laboratory.

[0056] Example 2: Real-time quantitative PCR

[0057] In this invention, the primers used for real-time quantitative PCR are shown in Table 1, the SYBR-Green amplification program is shown in Table 2, and the reaction system is shown in Table 3.

[0058] Table 1. Primer sequences used in the study

[0059]

[0060] Table 2. Dye-based qPCR amplification program

[0061]

[0062] Table 3. Dye-based reaction system

[0063]

[0064] In this invention, GraphPad-Prism 8.3.0 software is used for data analysis.

[0065] Example 2: Screening for monomeric components with anti-Coxsackievirus activity from 16 breast milk components.

[0066] 2.1 Formulation of 16 Breast Milk Components

[0067] Based on the main components of breast milk, four protein solutions were prepared: osteopontin solution, milk fat globule membrane solution, whey protein solution, and lactoferrin solution. Eight carbohydrate compound solutions were prepared: 2'-fucosylated lactose solution, 3'-sialylated lactose solution, lactose-N-neotetrasaccharide solution, lactose-N-tetrasaccharide solution, 3'-fucosylated lactose solution, 6'-sialylated lactose solution, galactooligosaccharide solution, and fructooligosaccharide solution. Four vitamin compound solutions were prepared: vitamin B1 solution, vitamin B2 solution, vitamin D2 solution, and vitamin D3 solution.

[0068] The initial concentration of the osteopontin solution was 5 mg / ml, the solute was osteopontin (Arla), and the solvent was PBS.

[0069] The initial concentration of the milk fat globule membrane solution was 5 mg / ml, with milk fat globule membrane (Glanbia) as the solute and PBS as the solvent.

[0070] The initial concentration of the whey protein solution was 5 mg / ml, with whey protein (Fonterra) as the solute and PBS as the solvent.

[0071] The initial concentration of the lactoferrin solution was 5 mg / ml, with lactoferrin as the solute and PBS as the solvent.

[0072] The initial concentration of the 2'-fucosylated lactose solution was 10 mg / ml, with 2'-fucosylated lactose (Glycome) as the solute and PBS as the solvent.

[0073] The initial concentration of the 3'-sialactose solution was 10 mg / ml, with 3'-sialactose (Glycome) as the solute and PBS as the solvent.

[0074] The initial concentration of the lactose-N-neotetrasaccharide solution was 10 mg / ml, with lactose-N-neotetrasaccharide (Glycome) as the solute and PBS as the solvent.

[0075] The initial concentration of the lactose-N-tetrasaccharide solution was 10 mg / ml, with lactose-N-tetrasaccharide (Glycome) as the solute and PBS as the solvent.

[0076] The initial concentration of the 3'-fucosylated lactose solution was 10 mg / ml, with 3'-fucosylated lactose (Glycome) as the solute and PBS as the solvent.

[0077] The initial concentration of the 6' sialic acid lactose solution was 10 mg / ml, with 6' sialic acid lactose (Glycome) as the solute and PBS as the solvent.

[0078] The initial concentration of the galactooligosaccharide solution was 10 mg / ml, with galactooligosaccharide (Quantum High-tech) as the solute and PBS as the solvent.

[0079] The initial concentration of the fructooligosaccharide solution was 10 mg / ml, the solute was fructooligosaccharide (Meiji), and the solvent was PBS;

[0080] The initial concentration of vitamin B2 was 100 ug / ml, the solute was vitamin B2 (provided by Biostime Group), and the solvent was PBS;

[0081] The initial concentration of vitamin B1 was 1 mg / ml, with vitamin B1 (provided by Health & Happiness Group) as the solute and PBS as the solvent.

[0082] The initial concentration of vitamin D2 was 1 mg / ml, with vitamin D2 (provided by Biostime Group) as the solute and PBS as the solvent.

[0083] The initial concentration of vitamin D3 was 1 mg / ml, with vitamin D3 (provided by Biostime Group) as the solute and PBS as the solvent.

[0084] PBS was purchased from Cytiv, with catalog number SH30256.01.

[0085] 2.2 Screening for potential anti-coxsackievirus drugs from 16 breast milk components using Coxsackievirus A9 strain BUCT01.

[0086] 2.5 × 10⁶ cells were seeded in 96-well cell plates. 4 RD cells were cultured for 24 hours and then infected with the CV-A9 strain BUCT01 to achieve an MOI of 0.001 in each well. Simultaneously, 50 μL of each of the 16 solutions prepared in 2.1 were added to each well. An equal volume of DMEM medium was added as a control. After culturing for another 24 hours, cytopathic effects were observed under a microscope. RNA was extracted from the cells in each well, and qRT-PCR was used to determine viral replication and the expression of the internal reference gene GAPDH. In the absence of significant cytotoxicity, a viral replication inhibition rate of over 90% compared to the control group was considered a potential anti-Coxsackievirus drug.

[0087] The results of the relative gene expression levels of the CV-A9 strain BUCT01 in cells from 16 breast milk component solutions are as follows: Figure 1 As shown, Figure 1 The results showed that the milk fat globule membrane, lactoferrin, and 2'-fucolactose inhibited the CV-A9 strain BUCT01 by more than 90%.

[0088] The inhibition rate of 2'-fucosylated lactose on the CV-A9 strain BUCT01 in cells is as follows: Figure 2 As shown, Figure 2 The results showed that 10 mg / ml of 2'-fucosylated lactose and 5 mg / ml of 2'-fucosylated lactose inhibited the CV-A9 strain BUCT01 by 99.97% and 99.19%, respectively.

[0089] The viral inhibition rate is calculated based on the Ct value and is represented by I.

[0090] The formula for calculating the inhibition rate is:

[0091] A = 2^(Ct) GAPDH -Ct CV-A9 );

[0092] I 加药处理组 (%) = 1 - (A) 加药处理组 / A 对照组 )×100%.

[0093] After 48 hours of culture without virus, the morphology of RD cells without drugs is as follows: Figure 7 As shown, Figure 7 The results showed that the cells grew well and maintained their morphology; 48 hours after infection with the CV-A9 strain BUCT01 (multiplicity of infection 0.001), the morphology of RD cells without medication was as follows: Figure 8 As shown, Figure 8 The results showed that CV-A9 infection resulted in sparse cell density and significant cell death. After culturing RD cells for 48 hours with a final concentration of 10 mg / ml of 2'-fucosylated lactose and an infection multiplicity of 0.001 for the CV-A9 strain BUCT01, the morphology was as follows: Figure 9 As shown, Figure 9 The results showed that the cells grew well, maintained their morphology, and did not show any lesions.

[0094] Example 3: Antiviral drug EC 50 and CC 50 Measurement

[0095] Seed 2.5 × 10⁶ cells in a 96-well cell plate 4 RD cells were infected with the CV-A9 strain BUCT01 24 hours later, with an MOI of 0.001 in each well. Simultaneously, 2'-fucosylated lactose was added to achieve concentrations of 20 mg / ml, 10 mg / ml, 5 mg / ml, 2.5 mg / ml, 1.25 mg / ml, 6.25 mg / ml, 3.125 mg / ml, 1.5625 mg / ml, 0.78125 mg / ml, and 0.390625 mg / ml. On day 2, cytopathic effects were observed under a microscope. RNA was extracted from wells without obvious cytopathic effects, and viral replication and the expression of the internal reference gene GAPDH were measured using real-time quantitative PCR as described in Example 2. The experiment was repeated three times, with three wells per concentration per replicate.

[0096] EC 50 This refers to the drug concentration that can effectively inhibit 50% of cell infection by the virus. The lower the value, the better the inhibitory effect on the virus.

[0097] CC 50 It is the drug concentration at which 50% of cells become diseased; the higher the value, the lower the toxicity to cells.

[0098] SI: Selectivity Index, represented by CC 50 With EC 50 The higher the ratio, the higher the likelihood of it becoming a drug.

[0099] 2'-fucosylated lactose on the ECG of CV-A9 strain BUCT01 50 and CC 50 The measurement results are as follows Figure 3 As shown, Figure 3 The left ordinate represents the inhibition rate of 2'-fucosylated lactose against CV-A9 strain BUCT01, the right ordinate represents the toxicity of 2'-fucosylated lactose against human rhabdomyosarcoma cells, and the x-axis represents the 2'-fucosylated lactose concentration log. 10 (mg / ml). Figure 3 The results showed that 2'-fucosylated lactose had an effect on the ECG of CV-A9 strain BUCT01. 50 =0.9716mg / ml, CC 50 >20 mg / ml. 2'-Fucosyllactose showed significant antiviral effects at concentrations of 20-5 mg / ml (inhibition rate of 99.9%-99.1%), without a significant increase in cytotoxicity. This suggests that 2'-fucosyllactose is a potent inhibitor of CV-A9 infection.

[0100] Example 4: Dosing Time Experiment

[0101] 2.5 × 10⁶ cells were seeded into 24-well cell plates. 5 RD cells were infected with the CV-A9 strain BUCT01 after 24 hours, with an MOI of 0.001 in each well. 2'-fucosylated lactose was added at a concentration of 10 mg / ml at each well during the entire infection cycle (at virus addition and 2 hours after virus addition incubation), before cell entry (at virus addition), and after cell entry (2 hours after virus addition incubation). After 24 hours of further culture, cytopathic effects were observed under a microscope, and RNA was extracted from the cells in the culture wells. qRT-PCR was used to determine viral replication in the cells and supernatant, as well as the expression of the internal reference gene GAPDH. An equal volume of DMEM medium was added to the positive control group.

[0102] The results are as follows Figure 4 As shown. Figure 4 The bar charts, from left to right, show the relative mRNA expression levels of the virus throughout the entire infection cycle, before cell entry, and after post-cell entry following the addition of 10 mg / ml of 2'-fucosylated lactose. Figure 4 It can be seen that the relative expression level of viral mRNA during the entire infection cycle and before viral entry is lower than that after viral entry and in the positive control, indicating that 2'-fucosylation lactose mainly plays a role in the viral entry stage.

[0103] Example 5: Adsorption and internalization experiment

[0104] Adsorption experiment: 0.1 × 10⁻⁶ cells were seeded in a 12-well cell plate. 6RD cells were incubated for 24 hours after the virus strain BUCT01 was mixed with 2'-fucosylated lactose at a final concentration of 10 mg / ml. The MOI of BUCT01 was set to 5, and the cells were cultured at 4°C for 2 hours. RNA was then extracted from the culture wells, and the viral load and expression of the internal reference gene GAPDH were determined by qRT-PCR.

[0105] Internalization experiment: 0.1 × 10⁻⁶ cells were seeded in a 12-well cell plate. 6 RD cells were infected with the CV-A9 strain BUCT01 with an MOI of 5 after 24 hours. The cells were cultured at 4°C for 2 hours, followed by the addition of 2'-fucosylated lactose at a final concentration of 10 mg / ml. The cells were then transferred to 37°C and cultured for 1 hour. Proteinase K was added for 15 minutes to digest the uncoated and internalized virus on the cell surface. RNA was then extracted from the culture wells, and the viral load and expression of the internal reference gene GAPDH were determined by qRT-PCR. An equal volume of DMEM medium was added to the positive control group.

[0106] The results are as follows Figure 5 As shown. Figure 5 In the figure, A represents the relative mRNA expression level of the virus in the adsorption experiment. Figure 5 B represents the relative mRNA expression level of the virus in the internalization experiment. Figure 5 It can be seen that the relative mRNA expression levels of the virus in the adsorption and internalization experiments were significantly lower than those in the positive control, indicating that 2'-fucosylation lactose plays an inhibitory role in both the adsorption and internalization stages of the virus.

[0107] Example 6: Exploring the specific effects of the drug

[0108] Seed 2.5 × 10⁶ cells in a 24-well cell plate 5 RD cells were infected with CV-A9 strain BUCT01 24 hours later, so that the MOI of CV-A9 strain BUCT01 was 5. 2'-fucosylated lactose was added at a final concentration of 10 mg / ml at different time points. 6 hours after infection, cellular RNA was extracted from the culture wells, and the viral content in the cells and the expression of the intracellular reference gene GAPDH were determined by qRT-PCR.

[0109] The specific timing of drug administration is as follows: 1 hour before infection, during infection, 30 minutes after infection, 1 hour after infection, and 2 hours after infection. The positive control group was given an equal volume of DMEM culture medium.

[0110] The results are as follows Figure 6 As shown, Figure 6The bar charts from left to right represent the times when 2'-fucosylated lactose was added: 1 hour before infection, at infection, 30 minutes after infection, 1 hour after infection, and 2 hours after infection, with a positive control. Figure 6 The bar chart shows that the relative expression level of viral mRNA in the treatment groups with 2'-fucosylated lactose added 1 hour before infection and at infection time was significantly lower than that in the experimental group and positive control group after infection. Figure 6 The results showed that 2'-fucosyllactose mainly functions before and during infection, and pre-incubation before infection can prevent infection, suggesting that 2'-fucosyllactose can be used as an effective drug to prevent CV-A9 infection.

[0111] In this invention, the inventors found that adding 2'-fucosylated lactose to RD cells infected with Coxsackievirus A9 strain BUCT01 resulted in a good dose-dependent anti-Coxsackievirus effect and low cytotoxicity. Currently, there are no specific drugs for Coxsackievirus A9 or other similar Coxsackieviruses or other Enterovirus B groups. This invention has important reference value for the future clinical use of Coxsackievirus A9 or other similar Coxsackieviruses or other Enterovirus B groups.

[0112] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. 2' The use of fucoidyl lactose or a pharmaceutically acceptable salt thereof, or a composition containing any one of them, characterized in that: The application is the following (a) and / or (b) and / or (c): (a) The use of 2'-fucosylated lactose or a pharmaceutically acceptable salt thereof or a composition containing any one thereof in the preparation of a medicament for the prevention of Coxsackievirus A9 infection; (b) The use of 2'-fucosylated lactose or a pharmaceutically acceptable salt thereof or a composition containing any one thereof in the preparation of a medicament for treating Coxsackievirus A9 infection; (c) Use of 2'-fucosylated lactose or a pharmaceutically acceptable salt thereof or a composition containing any one thereof in the preparation of an inhibitor of Coxsackievirus A9.

2. The application according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the complete genome of Coxsackievirus A9 is SEQ ID No.

2.

3. The application according to claim 2, characterized in that: The complete genome nucleotide sequence of the Coxsackievirus A9 is SEQ ID No.

1.

4. The application according to any one of claims 1-3, characterized in that: The drug or Coxsackievirus A9 inhibitor prevents and / or treats Coxsackievirus A9 infection by inhibiting the adsorption of Coxsackievirus A9 to host cells.

5. The application according to any one of claims 1-3, characterized in that: The drug or Coxsackievirus A9 inhibitor prevents and / or treats Coxsackievirus A9 infection by inhibiting the entry of Coxsackievirus A9 into host cells.

6. The application according to any one of claims 1-3, characterized in that: The drug or Coxsackievirus A9 inhibitor inhibits the proliferation of Coxsackievirus A9.

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