阅读说明：本技术 瑞德西韦（Remdesivir）在制备抗牛副流感病毒3型药物中的应用 (Application of Reddesivir (Remdesivir) in preparation of anti-bovine parainfluenza virus type 3 medicine ) 是由 程凯慧 于志君 杨宏军 楚会萌 朱彤 任亚初 于 2021-03-30 设计创作，主要内容包括：本发明涉及医药技术领域,具体涉及瑞德西韦在制备抗牛副流感病毒3型药物中的应用,所述抗BPIV3包括以下一种或多种作用：抑制BPIV3病毒增殖；灭活BPIV3病毒；阻止BPIV3对细胞的吸附作用；阻断BPIV3在细胞内的复制。本发明首次发现化合物瑞德西韦能够有效抑制BPIV3的增殖,且对细胞的毒性较小,经实验证明,瑞德西韦在体外实验MDBK细胞模型上能够有效抑制和杀灭BPIV3,能够有效抑制BPIV3的入侵和复制,可作为一类新的抗BPIV3的药物,为瑞德西韦开辟了新的药物用途,也为开发高效特异的抗BPIV3药物奠定实验基础并提供新的视野。(The invention relates to the technical field of medicines, in particular to application of Reidcvir in preparing an anti-bovine parainfluenza virus type 3 medicine, wherein the anti-BPIV 3 has one or more of the following effects: inhibiting BPIV3 virus proliferation; inactivating the BPIV3 virus; preventing the adsorption of BPIV3 to cells; blocking the replication of BPIV3 in the cell. The invention discovers for the first time that the compound Reidexi Wei can effectively inhibit the proliferation of BPIV3 and has low toxicity to cells, and experiments prove that Reidexi Wei can effectively inhibit and kill BPIV3 on an MDBK cell model in vitro experiments, can effectively inhibit the invasion and replication of BPIV3, can be used as a new anti-BPIV 3 drug, opens up new drug application for Reidexi Wei, also lays an experimental foundation for developing high-efficiency and specific anti-BPIV 3 drugs and provides a new visual field.)

1. Application of Reidesciclovir in preparing anti-BPIV 3 medicine.

2. The use of claim 1, wherein the anti-BPIV 3 comprises one or more of the following effects:

(1) inhibiting BPIV3 virus proliferation;

(2) inactivating the BPIV3 virus;

(3) preventing the adsorption of BPIV3 to cells;

(4) blocking the replication of BPIV3 in the cell.

3. The use according to claim 1, wherein the anti-BPIV 3 medicament has a medicament concentration of ridciclovir of not less than 0.39 μ M.

4. A pharmaceutical formulation against BPIV3, characterized in that it consists of reidesavir and at least one other non-pharmaceutically active ingredient.

5. The pharmaceutical formulation of claim 4 resistant to BPIV3 wherein the pharmaceutical concentration of Reidesciclovir is not less than half the effective concentration;

preferably, the median effective concentration of Reidesciclovir to BPIV3 is 0.39. mu.M.

6. The pharmaceutical formulation of claim 4, wherein the non-pharmaceutically active ingredient comprises a pharmaceutically acceptable carrier, excipient, and/or diluent.

7. The pharmaceutical formulation of claim 4, wherein the non-pharmaceutically active ingredient comprises:

pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids;

pharmaceutically harmless salt (preferably sodium chloride), flavoring agent, vitamin (preferably vitamin A or vitamin E, tocopherol or provitamin), antioxidant (preferably ascorbic acid), and stabilizer and/or antiseptic.

8. The pharmaceutical formulation of claim 4 that is resistant to BPIV3, wherein the pharmaceutical formulation is administered in a dosage form comprising: liquid dosage forms, solid dosage forms, external preparations and sprays;

preferably, the following dosage forms are included: true solutions, colloids, microparticles, emulsion, suspension, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, and liniment.

9. A pharmaceutical composition against BPIV3, characterized in that it consists of reidesavir and at least one other pharmaceutically active ingredient.

10. The pharmaceutical composition of claim 9 against BPIV3, wherein the further pharmaceutically active ingredient comprises a substance that inhibits and/or kills BPIV3 or assists in inhibiting and/or killing BPIV 3.

Technical Field

The invention relates to the technical field of medicines, in particular to application of Reidesvir in preparation of an anti-bovine parainfluenza virus type 3 medicine.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Bovine parainfluenza virus type 3 (BPIV 3) belongs to the family of paramyxoviridae and the genus pneumovirus, is the most important pathogen of Bovine respiratory syndrome (BRDC), is clinically characterized by fever, cough, asthma, anorexia, increased eye and nose secretions, few accompanied by diarrhea, and finally shows pneumonia, and can seriously cause abortion. Currently, BPIV3 infection is distributed worldwide, causing serious economic losses to the world cattle industry. In 1959 the virus was first isolated in the united states and subsequently in countries such as france, former soviet union, japan, danish, canada, australia, panama, italy, argentina and korea, the BPIV3 was later reported in our country.

BPIV3 is one of the important pathogens responsible for upper respiratory tract infections in calves and adult cattle, and members of the BPIV3 genus also include Human parainfluenza virus type 3 (HPIV 3) and Sendai virus (SeV). Under natural conditions, the disease only infects cattle, most of which are bred in barn, sick cattle and cattle with virus are main infection sources, and susceptible cattle are infected through air-droplets via respiratory tracts due to contact with the cattle with toxin expelling, and intrauterine infection can also occur. Viruses have been found in bull semen and cow reproductive tract and may cause infertility. In addition, BPIV3 was isolated by researchers from dairy cow clinical mastitis. The disease is common in late autumn and winter, and the cattle infected with BPIV3 are often damaged by respiratory epithelium to cause the defense capability of respiratory mucosa to be reduced and cause immunosuppression, so that the sick cattle infected with BPIV3 are often susceptible to secondary infection with serious bacterial or mycoplasma diseases to cause serious pneumonia, and the death rate is greatly increased.

Reidesivir (Remdesivir) is a nucleoside analog, has antiviral activity, and has certain inhibitory effects on filoviruses (Ebola, etc.), arenaviruses (Lassa fever virus, etc.), coronaviruses (SARS, MERS, etc.) in existing laboratory studies. 2016, Nature articles published by USAMRIID and Gilidard cooperation report the action mechanism of the Redexilvir, rhesus monkey PK/PD and in vivo Ebola virus resistant effect, and show that the medicament has high in-vitro MERS virus inhibition rate. However, no report on the application of the composition in preventing or treating bovine parainfluenza virus type 3 exists so far.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of Remdesivir (Remdesivir) in preparing the anti-BPIV 3 medicament, and the Remdesivir (Remdesivir) is proved to be capable of effectively inhibiting the proliferation of BPIV3 for the first time and has low toxicity to cells, so the Remdesivir (Remdesivir) has a prospect of being developed into the anti-BPIV 3 medicament.

In order to solve the technical problems in the prior art, the invention aims to provide an application of Reidesciclovir in preparing an anti-BPIV 3 medicament.

Specifically, the technical scheme of the invention is as follows:

in a first aspect of the invention, the application of the Reidesciclovir in preparing the anti-BPIV 3 medicament is provided.

In a second aspect of the invention, there is provided a pharmaceutical formulation against BPIV3, said pharmaceutical formulation consisting of reidesavir (Remdesivir) in combination with at least one other non-pharmaceutically active ingredient.

In a third aspect of the present invention, there is provided a pharmaceutical composition against BPIV3, said pharmaceutical composition consisting of reidesavir (Remdesivir) in combination with at least one other pharmaceutically active ingredient.

The specific embodiment of the invention has the following beneficial effects:

the invention discovers for the first time that the compound Remdesivir (Remdesivir) can effectively inhibit the proliferation of BPIV3, has relatively low toxicity to cells, and experiments prove that the Remdesivir (Remdesivir) can inhibit BPIV3 virus; inactivating the BPIV3 virus; preventing the adsorption of BPIV3 to cells; block BPIV3 replication in cells;

the median cytotoxic concentration (CC50) of Reidesciclovir (Remdesivir) on MDBK cells was 12.5. mu.M, while the median effective concentration (EC50) on BPIV3 virus was 0.39. mu.M; the therapeutic index of Remdesivir (Remdesivir) to BPIV3 is 32, which shows that the Remdesivir (Remdesivir) has the prospect of being developed into anti-BPIV 3 drugs, opens up new drug application for Remdesivir (Remdesivir), also lays an experimental foundation for developing high-efficiency and specific anti-BPIV 3 drugs and provides a new visual field.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the effect of Remdesivir (Remdesivir) of the present invention on BPIV3 injured cells;

wherein: wherein FIG. 1A is a viral control group; FIG. 1B is a set of MDBK normal cells; FIG. 1C is a drug test group of infected cells (using 0.39. mu.M Remdesivir);

FIG. 2 is a graph of the half cytotoxic concentration (CC50) of Reddesivir (Remdesivir) against MDBK cells in example 2;

FIG. 3 is a graph of the median effective concentration (EC50) of Reddeevir (Remdesivir) versus BPIV3 in example 3;

FIG. 4 is a graph of the effect of Reidesciclovir (Remdesivir) administration at various time points on BPIV3 inhibition in example 4;

FIG. 5 is a graph showing the direct killing effect of Reddevir (Remdesivir) on BPIV3 in example 5.

FIG. 6 is a graph showing the effect of example 5 on the blocking of adsorption of BPIV3 by Remdesivir (Remdesivir).

FIG. 7 is a graph of the effect of example 5 Reddesivir (Remdesivir) on the blockade of BPIV3 replication.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. The following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the contents thereof. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

As discussed in the background, there has been no report in the prior art of the use of renciclovir (Remdesivir) for the prevention and treatment of BPIV3, and in view of this, the present invention provides the use of renciclovir (Remdesivir) in the preparation of anti-BPIV 3 drugs.

It should be noted that the application is disclosed for the first time, and is different from the known clinical application, and the structural formula of the Reidesciclovir is as follows:

in one embodiment of the invention, the application of the Reidesciclovir in preparing the anti-BPIV 3 medicament is provided, wherein the anti-BPIV 3 comprises the prevention and/or treatment of BPIV3 related diseases;

preferably, the anti-BPIV 3 includes one or more of the following effects:

(1) inhibition of BPIV3 virus;

(2) inactivating the BPIV3 virus;

(3) preventing the adsorption of BPIV3 to cells;

(4) block BPIV3 replication in cells;

according to the present invention, the concept of "prevention and/or treatment" means any measure suitable for treating a BPIV 3-related disease, or to prophylactically treat such an manifested disease or manifested symptoms, or to avoid recurrence of such a disease, such as recurrence after the end of a treatment period or treatment of symptoms of an already-developed disease, or pre-interventional prevention or inhibition or reduction of the occurrence of such a disease or symptoms.

The anti-BPIV 3 medicine is a substance which has obvious inhibiting and killing effects on BPIV3 and has good effects on the direct killing, adsorption blocking and replication blocking of viruses.

As an alternative or in addition to other pharmaceutically active ingredients, reidesivir (Remdesivir) may also be used in combination with other non-pharmaceutically active ingredients.

In one embodiment of the present invention, there is provided a pharmaceutical formulation against BPIV3, said pharmaceutical formulation consisting of reidesavir (Remdesivir) in a pharmaceutical concentration not lower than half the effective concentration (EC50) in combination with at least one other non-pharmaceutically active ingredient;

preferably, the half effective concentration of Reddeevir (Remdesivir) to BPIV3 (EC50) is 0.39 μ M; the therapeutic index of Reddesivir (Remdesivir) to BPIV3 is 32;

preferably, the non-pharmaceutically active ingredient includes, but is not limited to, a pharmaceutically acceptable carrier, excipient, and/or diluent.

Further preferably, the non-pharmaceutically active ingredients include: pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, pharmacologically harmless salts such as sodium chloride, flavoring agents, vitamins such as vitamin a or vitamin E, tocopherols or provitamins, antioxidants such as ascorbic acid, and stabilizers and/or preservatives for prolonging the use and shelf life of pharmaceutically active ingredients or formulations, and other common non-pharmaceutically active ingredients or adjuvants and additives known in the art, and mixtures thereof.

The pharmaceutical formulation may be administered in unit dosage form; the administration dosage form may be a conventional formulation form of Reidesvir (Remdesivir), or other feasible dosage forms, such as those skilled in the art can select a dosage form suitable for Reidesvir (Remdesivir) from conventional dosage forms by adding carriers, excipients, binders, diluents and the like compatible with Reidesvir (Remdesivir).

The conventional dosage form comprises: liquid dosage forms, solid dosage forms, external preparations and sprays; further, the preparation comprises the following dosage forms: true solutions, colloids, microparticles, emulsion, suspension, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, and liniment.

In one embodiment of the present invention, there is provided a pharmaceutical composition against BPIV3, consisting of reidesavir (Remdesivir) in combination with at least one other pharmaceutically active ingredient.

Preferably, the other pharmaceutically active ingredient comprises a substance having the ability to inhibit and/or kill BPIV3 or to assist in inhibiting and/or killing BPIV 3.

Preferably, the pharmaceutical concentration of the Reidesciclovir (Remdesivir) is not lower than half the effective concentration (EC 50);

further preferably, the half effective concentration of Reidesciclovir (Remdesivir) to BPIV3 (EC50) is 0.39 μ M; the therapeutic index of Reddesivir (Remdesivir) to BPIV3 was 32.

In a particular embodiment, when Reidesivir (Remdesivir) is used in combination with other drugs or active ingredients having inhibitory and/or killing properties or assisting in the inhibition and/or killing of BPIV3 for the same application as mentioned in the summary of the invention, the drug concentration may theoretically be lower than the half-effective concentration mentioned above, without excluding special exceptions.

According to the present invention, not only the use of Remdesivir (Remdesivir) for the preparation of anti-BPIV 3 drugs, but also the enhancement of this effect when administered in combination with at least one other pharmaceutically active ingredient is disclosed.

The technical solution of the present invention is further described with reference to the following specific examples.

It should be noted that the BPIV3 used in the examples of the present application was isolated from the research center for dairy cows of the academy of agricultural sciences of shandong province and was identified as belonging to the BPIV 3C type.

The specific separation and identification method is as follows: diluting fresh nasal swab with PBS, repeatedly freezing and thawing for 3 times, centrifuging at 5000 r/min for 5min, collecting supernatant, adding penicillin (200IU/mL) and streptomycin (100 μ g/mL), collecting 200 μ L supernatant, extracting BPIV3 virus genome RNA according to virus genome DNA/RNA extraction kit specification, reverse transcribing into cDNA, performing PCR amplification with BPIV3 specific primer, filtering and sterilizing the nasal swab liquid identified as positive by PCR with 0.22 μm filter membrane, collecting 1mL inoculated MDBK single layer cell, adsorbing at 37 deg.C for 1h, discarding, adding into DMEM culture medium containing 2% FBS, and culturing at 37 deg.C and 5% CO for 5min₂Culturing in a cell culture box, observing cytopathic condition every 12h, and continuously observing for 3 d. Freezing and thawing cells with pathological Changes (CPE) for 3 times repeatedly, collecting cell virus liquid, identifying whether the obtained cell virus liquid is single BPIV3 infection or not, and obtaining the cell virus liquid with single BPIV3 infection by using a plaque purification method if the cell virus liquid is not single virus infection. The isolate was identified to be of BPIV 3C type by phylogenetic tree analysis of BPIV 3.

Example 1

Virus TCID₅₀Measurement of (2)

MDBK cells (stored in Dairy research center of Oncology institute of agriculture, Shandong province) were digested at 1X 10 per well⁵Cell density of one/mL was seeded into 96-well cell culture plates and placed at 37 ℃ in 5% CO₂After culturing the cells in the cell culture chamber of (1) to form a monolayer of cells, the cell growth medium in the wells was discarded, and BPIV3 was continuously diluted 10-fold with virus dilutions (10 dilutions, respectively)^-1～10^-10) Inoculating to a 96-well plate full of monolayer cells, each well having a volume of 100 μ L, placing at 37 deg.C and 5% CO₂The incubator of (1) was continued, and the CPE of the cells was observed day by day, andfine record cytopathic well number. And setting a normal cell control group and a blank control group at the same time, setting 8 repeats in each group, and judging the result when the cytopathic effect is not continued. The cell lesion hole is a cell hole corresponding to the above cell lesion, and virus TCID is calculated by Karber method₅₀。

TABLE 1 TCID₅₀ of BPIV3

Note: TCID₅₀Tissue culture infectious dose, also known as 50% Tissue cell infectious dose; i.e., the amount of virus required to cause half of the cytopathic effect or death (CPE) in a well or tube in culture.

As a result: morphological observation under a microscope shows that virus diluents with different concentrations all cause cytopathic effect when 36 hours elapse, the refractive index of cells changes, the monolayer structure is destroyed, cells are subjected to round shrinkage necrosis and gradually take the shape of a net and form vacuoles, some cells are cracked and fall off into fragments, cytopathic effect of each hole is not continued after 72 hours, the number of CPE holes with different concentrations is counted, the CPE ratio with different concentrations is calculated, and the TCID of BPIV3 is calculated according to a Karber method₅₀The value:

LgTCID₅₀＝L-D(S-0.5)

(L: logarithm of highest dilution; D: difference between logarithm of dilutions; sum of S-positive well ratios)

LgTCID₅₀＝L-D(S-0.5)＝-1-1×(5.75-0.5)＝-3.75

TCID₅₀＝10^-6.25/0.1mL

I.e. diluting the virus 10^6.25Inoculation with 100. mu.L resulted in 50% of the cells being diseased.

Example 2

Toxicity test of Reidesciclovir (Remdesivir) on MDBK cells:

MDBK cells are susceptible cells to BPIV 3. Therefore, the cytotoxicity of the Reidesciclovir (Remdesivir) on MDBK cells is firstly detected, and the specific experimental steps are as follows:

(1) mu.L of cells (MDBK 1X 10) were seeded in 96-well plates⁴One/hole).

(2) After incubation to MDBK monolayer, the next dosing analysis was performed. Media was discarded and 100 μ L of 2% FBS DMEM containing different drug concentrations were added to each well, 3 replicates for each concentration. At the same time, control wells: add 100. mu.L of 2% FBS DMEM medium. Zero setting hole: cells were not plated.

(3) At 37 ℃ 5% CO₂After culturing for 48h under the condition, the OD value at 450nm is measured by an enzyme-labeling instrument according to the instruction of a CCK-8 kit.

(4)37℃，5％CO₂After further incubation for 2h under these conditions, the absorbance was measured at 450 nm. A450 nm of normal growing cells was set as 100% cell control.

(5) Data were analyzed and half the Cytotoxic Concentration (CC) of Reddesivir (Remdesivir) was calculated using GraphPad Prism5₅₀) The value is obtained. The results are shown in FIG. 2.

As a result: the Remdesivir (Remdesivir) has a dose-dependent relationship, namely, the cell pathological changes are obvious along with the increase of the concentration of the medicine. Through statistical analysis, the half poisoning concentration of the Reidesciclovir (Remdesivir) is determined to be 12.5 mu M.

Example 3

Inhibition of BPIV3 by reidesavir (Remdesivir):

(1) 1X 10 inoculations in each well of a 96-well plate⁴MDBK cells, 37 ℃, 5% CO₂Culturing in an incubator overnight;

(2) the medium was discarded and 100. mu.L of 1000TCID was added to each well₅₀BPIV3 dilutions (2% FBS DMEM cells after confluency with viral dilutions, 25 μ M initial concentration, two-fold concentration gradient dilution dosing, 5% CO₂Culturing in an incubator;

(3) after 48h, the OD at 450nm was measured with a microplate reader, following the instructions of the CCK-8 kit.

(4) The data were analyzed and the viral inhibition ratio (%) (drug-treated D450nm value-virus control D450nm value)/(plusNormal cell control group D450nm value-virus control group D450nm value). times.100%, and half Effective Concentration (EC) of compound obtained by GraphPad Prism5 software₅₀) The value is obtained. The results are shown in FIG. 3. Then according to the formula TI ═ CC₅₀/EC₅₀And calculating the corresponding therapeutic index TI value.

As a result: the effective inhibition rate of the medicament on the BPIV3 can be calculated by detecting the cell viability through a CCK-8 kit. From the results, the effective inhibition rate of the Remdesivir (Remdesivir) in a safe concentration range is increased along with the increase of the drug concentration, and the effective inhibition rate is in a certain dose-effect relationship. Half Effective Concentration (EC) of BPIV3 by analytical software₅₀) It was 0.69. mu.M. The therapeutic index of Reddesivir (Remdesivir) to BPIV3 was 32.

Example 4

Preliminary study of mechanism of action

The action period of the Reindesivir (Remdesivir) is preliminarily judged by adding the test compound to the MDBK cells inoculated with the BPIV3 through different administration time, namely corresponding time points of first administration and then infection of the virus (before 0h), first infection of the virus and then administration (after 0h), and simultaneous addition of the virus and the drug to the cells (0 h). The specific experimental steps are as follows:

(1) 1X 10 inoculations in each well of a 96-well plate⁴MDBK cells, 37 ℃, 5% CO₂Culturing in an incubator.

(2) According to the measured pharmacodynamic evaluation result of the related medicine, the concentration of the medicine required by the experiment is determined, and the medicine is diluted to the required concentration by using a maintenance medium.

(3) After overnight incubation, the cell supernatants from the second three duplicate wells of the 96-well plate were aspirated and the cells were washed 2 times with phosphate buffer. Then 50. mu.L of the drug to be tested was added, and the time was recorded as-2 h.

(4) After 2h, the cell supernatants from the other wells were aspirated off, and diluted BPIV3 was added to each well in columns 2-11 at a volume of 50. mu.L per well. At the same time, 50. mu.L of the corresponding analyte was added to the three duplicate wells in column 3, which was recorded as 0 h.

(5) And adding corresponding compounds to be detected into the three compound holes in the next row at regular intervals, and marking the corresponding time. MDBK cells from column 11 were used as virus control.

(6) After 48 hours of incubation, OD measurements were performed. The data were analyzed and concluded, the results of which are shown in fig. 4.

As a result: according to the analysis of the administration experiment results at different time points, the Reindesivir (Remdesivir) has obvious inhibition effect on viruses when the viruses infect cells for-2 h, 0h, 2h, 4h, 6h and 8 h.

Example 5

Effect of Compound addition at different times on BPIV3 replication

In vitro antiviral inhibition test is carried out on Remdesivir (Remdesivir) by adopting 3 different action modes of firstly adding medicine and then adding virus, firstly adding virus and then adding medicine, and pre-acting medicine and virus.

(1) Direct killing effect of medicine on virus

Equal amount of 1000TCID₅₀Mixing the virus solution with the medicinal diluent at different concentrations, and standing at 37 deg.C with 5% CO₂Pre-acting in an incubator for 4h, adding into a 96-well cell culture plate with a monolayer, allowing each liquid medicine to have a gradient of 100 μ L/well, acting in the incubator for 2h, discarding the supernatant, and adding cell maintenance liquid to continue culturing. The test simultaneously sets a normal cell control group, a virus control group and a blank control group, each concentration is set to be 3 times, cell viability detection is carried out for 48 hours, and GraphPad Prism5 software is used for obtaining EC of the compound₅₀。

As a result: the effect of Reddesivir (Remdesivir) on BPIV3 is shown in FIG. 5 by analytical software in a pre-treatment dosing regimen of Reddesivir (Remdesivir) with BPIV 3. As can be seen from fig. 5, in this formula, remidesivir (Remdesivir) shows an inhibitory effect on BPIV3 in a safe concentration range, indicating that remidesivir (Remdesivir) has a certain direct inactivation effect on BPIV 3.

(2) Blocking effect of drug on adsorption of BPIV3

At a rate of 1X 10 per hole⁴Cell density of cells digested cells were inoculatedPlanting into a pore plate, removing supernatant after monolayer cells grow, adding drug diluents with different concentrations into a 96-pore cell culture plate with a monolayer growth gradient of 100 mu L/pore, performing pre-action in an incubator for 4h, removing supernatant, washing twice with PBS, adding 1000TCID with equal amount, and collecting supernatant₅₀Placing the virus liquid at 37 ℃ and 5% CO₂Culturing in an incubator. The test is simultaneously provided with a normal cell control group, a virus control group and a blank control group, each concentration is set for 3 times, cell activity detection is carried out after 48 hours, and the antiviral effective rate of the drugs with different concentrations under the action mode is calculated.

As a result: the effect of the Reddesivir (Remdesivir) on the BPIV3 is shown in FIG. 6 through analysis software, and the result shows that the effective inhibition rate of 0.78 muM and above on the BPIV3 can reach 100% in a safe concentration range, which indicates that the Reddesivir (Remdesivir) can prevent the adsorption of the BPIV3 on cells.

(3) Blockade of BPIV3 replication by drugs

At a rate of 1X 10 per hole⁴Cell density digested cells were seeded into well plates, supernatant discarded after monolayer growth, and an equivalent amount of 1000TCID was added₅₀Adding virus solution into 96-well cell culture plate, and standing at 37 deg.C under 5% CO₂Pre-acting in an incubator for 2h, removing supernatant, washing cells for 2 times with PBS, adding drug diluents with different concentrations, each drug solution gradient being 100 μ L/well, setting a normal cell control group, a virus control group and a blank control group at the same time in the test, setting 3 repeats for each concentration, placing at 37 ℃ and 5% CO₂Culturing in an incubator, detecting cell viability after 48h, analyzing data and obtaining a conclusion.

As a result: the effect of the Reidesciclovir (Remdesivir) on the replication inhibition effect of BPIV3 is shown in FIG. 7 through analysis software, and the result shows that the effective inhibition rate of 0.78 μ M and above on BPIV3 can reach 100% in a safe concentration range, which indicates that the Reidesciclovir (Remdesivir) can effectively block the replication of BPIV3 in MDBK cells.

In the application embodiment of the invention, the bovine kidney cells (MDBK) are used as a carrier, and 3 different action modes of adding medicine first and then adding virus, adding medicine first and then adding medicine, and pre-acting virus and then adding medicine are adopted on a cytopathogenic model to carry out in-vitro antiviral inhibition research. The novel antiviral effect of the Remdesivir (Remdesivir) is found, and the Remdesivir (Remdesivir) has a certain inhibition effect on the BPIV 3.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.