Uses of BAY87-2243, Pseudorabies virus inhibitory drugs

Abstract

The present application belongs to the technical field of pseudorabies virus prevention and control, and discloses the use of BAY87-2243 to prepare a drug for inhibiting the proliferation of pseudorabies virus. The present invention first infects PK-15 with PRV-GD-WH strain, takes total cell RNA, and performs reverse transcription to obtain cDNA. The changes in the transcription levels of key genes of intracellular glycolysis are detected by SYBR Green fluorescence quantitative PCR. BAY87 is further used to act on HIF-1α to verify the regulatory effect of HIF-1α on glycolysis, and finally the intracellular viral load and TCID are detected by SYBR Green fluorescence quantitative PCR. 50 The inhibitory effect of BAY87-2243 on PRV proliferation is determined. At the same time, the present invention also provides a pseudorabies virus inhibitory drug.

Description

Technical Field

[0001] The present application relates to the technical field of pseudorabies virus prevention and control, and in particular to the use of BAY87-2243 and pseudorabies virus inhibitory drugs. Background Art

[0002] There are many types of herpesviruses, which seriously endanger the public health security of humans and animals around the world. Among them, pseudorabies virus (PRV) belongs to the alphaherpesviridae family and can infect and cause pseudorabies (PR). PRV has a wide range of hosts, including pigs, cattle, sheep, dogs, cats, mice, rabbits, etc. Pigs are the only natural host of PRV, and pigs at all stages are susceptible, resulting in abortion and stillbirth in pregnant sows, infertility in boars, encephalitis and high mortality in newborn piglets, breathing difficulties and growth stagnation in fattening pigs, causing huge economic losses. Vaccine immunization is the main means of preventing and controlling pseudorabies in pigs, but it can only prevent clinical symptoms in infected pigs and cannot completely block the establishment of PRV latent infection and excretion of toxins. It is worth noting that even if the pig herd is vaccinated for a long time, it still cannot completely prevent the spread and spread of PRV between different pigs, resulting in repeated occurrence and epidemic of pseudorabies, which seriously hinders the prevention and control and purification of the disease. At present, there is no drug or vaccine that can completely eliminate PRV in infected pigs, which increases the difficulty of preventing, controlling and purifying pseudorabies in pigs.

[0003] For virus replication and proliferation, the proliferation pathways are obviously different based on the differences in viruses. For example, the Foot-and-Mouth Disease and Emerging Disease Epidemiology Team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, revealed the molecular mechanism by which small RNA viruses such as foot-and-mouth disease virus (FMDV) promote the uptake of aspartate through the amino acid transporter SLC38A8, activate the PI3K / mTOR / p70S6K1 pathway, promote 40S ribosome activation, and thus promote efficient viral replication. Other studies have revealed that by detecting the promoter activity of IFN-β, NF-κB and ISRE and the transcription level of downstream molecule mRNA of the type I interferon (IFN-I) signaling pathway, it was found that USP18 molecules can promote the replication of CSFV. Some studies have also pointed out that cannabidiol (CBD) inhibits the replication of SARS-CoV-2 by upregulating the host IRE1αRNase endoplasmic reticulum (ER) stress response and interferon signaling pathway.

[0004] In short, since the viral replication pathway is very extensive and complex, in order to screen out the inhibition or activation of specific pathways for specific viruses to inhibit viral replication, extensive experiments are needed to find more suitable target reagents.

[0005] The problem to be solved in this scheme is: how to provide a new pseudorabies virus inhibitory drug. Summary of the invention

[0006] The purpose of the present invention is to provide a new use of BAY87-2243. The present invention first infects PK-15 with PRV-GD-WH strain, takes total cell RNA, and performs reverse transcription to obtain cDNA. The changes in the transcription levels of key genes of intracellular glycolysis are detected by SYBR Green fluorescence quantitative PCR. BAY87 is further used to act on HIF-1α to verify the regulatory effect of HIF-1α on glycolysis, and finally the intracellular viral load and TCID are detected by SYBR Green fluorescence quantitative PCR. 50 The inhibitory effect of BAY87-2243 on PRV proliferation was determined.

[0007] Meanwhile, the invention also provides a medicine.

[0008] Before describing the technical solution of the present invention in detail, the terms involved in the present invention are explained:

[0009] PRV: pseudorabies virus; MOI: multiplicity of infection; Dpi: duration of infection in days; Hpi: duration of infection in hours; PK-15: porcine kidney cells; Viral titer: virus titer; TCID 50 : half tissue culture infection dose; Western blot: protein immunoblotting; q-PCR: real-time quantitative PCR; HK2: hexokinase 2; MCT4: monocarboxylate transporter 4; ACTINβ: actin β.

[0010] To achieve the above objectives, the present application discloses the use of BAY87-2243 to prepare a drug for inhibiting the proliferation of pseudorabies virus.

[0011] Meanwhile, the invention also discloses a pseudorabies virus inhibitory drug containing BAY87-2243.

[0012] In the above-mentioned pseudorabies virus inhibitory drug, the drug is a preparation prepared by mixing BAY87-2243 with a carrier.

[0013] In the above-mentioned pseudorabies virus inhibitory drug, the pseudorabies virus inhibitory drug is a liquid preparation.

[0014] In the above-mentioned pseudorabies virus inhibitory drug, the pseudorabies virus inhibitory drug is an ointment, a paste, a solution, a perfumed water, an injection, an oral agent, or an aerosol.

[0015] The beneficial effects of this application are:

[0016] The present invention first infects PK-15 with PRV-GD-WH strain, takes total RNA from cells, and performs reverse transcription to obtain cDNA. The changes in the transcription levels of key genes of glycolysis in cells are detected by SYBR Green fluorescence quantitative PCR. BAY87 is further used to act on HIF-1α to verify the regulatory effect of HIF-1α on glycolysis, and finally the viral load in cells is detected by SYBR Green fluorescence quantitative PCR and TCID50 is used to determine the inhibitory effect of BAY87-2243 on PRV proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A This is a graph showing the up-regulation level of MCT4 after pseudorabies virus infection in PK-15 cells;

[0018] Figure 1B This is a graph showing the up-regulation level of HK2 after pseudorabies virus infection in PK-15 cells;

[0019] Figure 2 The results show that the protein level of HIF-1α is upregulated after pseudorabies virus infection in PK-15 cells;

[0020] Figure 3 The results show the effect of BAY87-2243 on the protein level of HIF-1α after PRV infection;

[0021] Figure 4A This is a graph showing the results of BAY87-2243 inhibiting the gene expression level of MCT4 by PRV;

[0022] Figure 4B This is a graph showing the results of BAY87-2243 inhibiting the gene expression level of HK2 by PRV;

[0023] Figure 5 The effect of BAY87-2243 on intracellular viral load;

[0024] Figure 6 TCID for BAY87-2243 against PRV 50 impact. DETAILED DESCRIPTION

[0025] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] Part I

[0027] Test materials

[0028] Experimental animals and viruses

[0029] The strain used in the experiment was PRV GD-WH, which was obtained from the Swine Disease Laboratory of the Institute of Animal Health, Guangdong Academy of Agricultural Sciences;

[0030] PK-15 cells were obtained from the Swine Disease Department of the Animal Health Institute of Guangdong Academy of Agricultural Sciences.

[0031] Main reagents and materials for the test

[0032] DMEM basic (Gibco), Australian fetal bovine serum, trypsin, BAY87-2243 (MCE), β-mercaptoethanol, TotalRNA Kit I R6834 (Omega Bio-Tek), Tissue DNA Kit D3396 (Omega Bio-Tek), BeyoECLMoon (ultra-sensitive ECL chemiluminescence kit) (Biyuntian), reverse transcription reagent: HiScript III RT SuperMixfor qPCR (+gDNA wiper) (Novozyme), TBS (20X), Tween20 (Bio Froxx), PVDF membrane, antibody ACTIN, HIF-1α.

[0033] Main solution preparation

[0034] SDS-PAGE electrophoresis buffer (10X): Tris base 15.15ɡ, glycine 72ɡ, SDS 5ɡ, deionized water to 500 ml;

[0035] Transfer buffer (10X): Tris base 15.15ɡ glycine 72ɡ, dilute to 500ml with deionized water;

[0036] Transfer buffer (1X): 50 ml transfer buffer (10X) dilute to 400 ml with deionized water and add 100 ml methanol;

[0037] PBS (10X): Na2HPO4 10.4ɡ, KH2PO4 1ɡ, NaCl 40ɡ, KCl 1ɡ, dilute to 500 ml with deionized water;

[0038] PBST (1X): Take 50 ml PBS (10X) and dilute to 500 ml with deionized water, add 0.1% Tween20 and mix well;

[0039] TBST: TBS (20X) diluted 20 times and added with 0.1% Tween20;

[0040] Blocking solution: 5% skim milk (weigh 2.5 g skim milk powder and dissolve in 50 ml PBST).

[0041] Part 2

[0042] 2.1 PRV infection upregulates glycolytic transcription levels

[0043] 2.1.1 Detection of the effect of PRV infection on glycolytic transcription levels in PK-15 cells

[0044] a. PK-15 cells were cultured at 1×10 6 Cells / well were inoculated into 6-well plates, 2 ml of 10% FBS DMEM was added to each well, and the plates were cultured in a constant temperature cell culture incubator at 37°C and 5% CO2.

[0045] b. When the cells grew to a density of 70-80%, the cells were infected with the PRV GD-WH strain at a multiplicity of infection of 1 (MOI=1), and a mock infection group (MOCK) was set up at the same time.

[0046] c. After continuous infection at 37°C for 4 / 8 / 12 hours (hpi: 4 / 8 / 12), discard the culture medium, rinse with PBS three times, collect the cell samples, and extract cellular RNA using the Total RAN Kit.

[0047] d. The extracted RNA was reverse transcribed using HiScript III RT SuperMix for qPCR reagent to obtain cDNA. The reverse transcription system and procedure are shown in Tables 1 and 2.

[0048] Table 1 Reverse transcription system and procedure 1

[0049]

[0050] Table 2 Reverse transcription system and procedure 2

[0051]

[0052] e. Using the cDNA obtained by reverse transcription as a template, SYBR Green fluorescence quantitative PCR was performed using Novazonase to detect changes in the transcription levels of key glycolytic genes in cells. The sequences of key glycolytic genes and their primers are shown in Table 3, the SYBR Green fluorescence quantitative reaction system is shown in Table 4, and the reaction procedure is shown in Table 5.

[0053] Table 3 Key genes of glycolysis and their primer sequences

[0054]

[0055] Table 4 Fluorescence quantitative reaction system

[0056] Reagents Dosage 2 × ChamQ Universal SYBR qPCR Mix 10 μL F-Primer 0.5 μL R-Primer 0.5 μL cDNA 1 μL <![CDATA[ddH2O]]> 8 μL

[0057] Table 5 Fluorescence quantitative reaction procedure

[0058]

[0059] f. GraphPad Prism 8 software was used to perform statistical analysis on the experimental results. Figure 1A and Figure 1B As shown, the results showed that PRV infection upregulated the mRNA levels of MCT4 and HK2 in PK-15 cells compared with the control group.

[0060] Figure 1A This is a graph showing the up-regulation level of MCT4 after pseudorabies virus infection in PK-15 cells;

[0061] Figure 1B This is a graph showing the up-regulation level of HK2 after pseudorabies virus infection in PK-15 cells;

[0062] Figure 1A and Figure 1B ns: no significant difference, *: P <0.05, significant difference; **: P <0.01, the difference is extremely significant; ***: P <0.001, the difference is extremely significant.

[0063] 2.2 PRV regulation of glycolysis depends on HIF-1α

[0064] 2.2.1 Detection of the effect of PRV infection on the activation level of HIF-1α

[0065] a. PK-15 cells were cultured at 1×10 6 Cells / well were inoculated into 6-well plates, 2 ml of 10% FBS DMEM was added to each well, and the plates were cultured in a constant temperature cell culture incubator at 37°C and 5% CO2.

[0066] b. When the cells grew to a density of 70-80%, the cells were infected with the PRV GD-WH strain at a multiplicity of infection of 1 (MOI=1), and a mock infection group (MOCK) was set up at the same time.

[0067] c. After 8 hours of infection at 37°C, discard the culture medium and rinse three times with PBS to collect the cell protein sample.

[0068] d. The effect of PRV on HIF-1α protein level was detected by Western blot;

[0069] HIF-1α is a key regulatory hub at the transcriptional level of glycolysis. The effect of PRV infection on HIF-1α was not detected. Protein samples of PK-15 cells were prepared after continuous PRV infection for 8 hours, and the changes in intracellular HIF-1α protein levels were detected by Western blot. The experimental results are as follows Figure 2 As shown, the results showed that PRV infection upregulated the protein level of HIF-1α.

[0070] 2.2.2 Detection of the inhibitory effect of BAY87-2243 on HIF-1α

[0071] a. PK-15 cells were pretreated with the HIF-1α activation inhibitor BAY87-2243 (200 nM) for 1 hour, and then infected with the PRV GD-WH strain at a multiplicity of infection of 1 (MOI=1). A mock infection group (MOCK) and a drug-treated control group were set up.

[0072] b. After infection at 37°C for 8 hours, discard the culture medium and rinse the cells three times with PBS to collect the cell protein samples.

[0073] c. The effect of PRV on HIF-1α protein level under the action of BAY87-2243 was detected by Western blot.

[0074] PK-15 cells were pretreated with BA87-2243 (200 nM), an effective inhibitor of HIF-1α, for 1 hour, and then infected with PRV GD-WH strain at a multiplicity of infection of 1 (MOI = 1) for 8 hours. Cell protein samples were collected and Western blot was used to detect changes in intracellular HIF-1α protein levels. The experimental results are shown in Figure 3 As shown, the results showed that PRV infection upregulated the protein level of HIF-1α, and BAY87-2243 could inhibit the activation of HIF-1α by PRV infection.

[0075] 2.2.3 Detection of the regulatory effect of BAY87 on glycolysis

[0076] a. PK-15 cells were pretreated with the HIF-1α activation inhibitor BAY87-2243 (200 nM) for 1 hour, and then infected with the PRV GD-WH strain at a multiplicity of infection of 1 (MOI=1). A mock infection group (MOCK) and a drug-treated control group were set up.

[0077] b. After 4 hours of continuous infection at 37°C, discard the culture medium, rinse the cells three times with PBS, collect the cell samples, and extract the cell RNA using the Total RAN Kit.

[0078] c. Use HiScript III RT SuperMix for qPCR reagent to reverse transcribe the extracted RNA to obtain cDNA.

[0079] d. Detect changes in transcription levels of key glycolytic genes in lung tissue by SYBR Green fluorescence quantitative PCR, using the same method as 2.1.1.e.

[0080] e. The experimental results were statistically analyzed using GraphPad Prism 8 software.

[0081] Further, the changes in glycolytic mRNA levels in pigs infected with PRV after the action of BAY87-2243 were detected. PK-15 cells were pretreated with BA87-2243 (200nM), an effective inhibitor of HIF-1α, for 1 hour, and then the cells were continuously infected with the PRV GD-WH strain with a multiplicity of infection of 1 (MOI=1) for 1 hour. Cell RNA samples were collected and reverse transcribed and fluorescent quantitative PCR was used to detect changes in mRNA levels of key glycolytic genes. The test results are as follows Figure 4A and Figure 4B As shown, the results showed that compared with the control group, PRV infection upregulated the mRNA levels of MCT4 and HK2 in PK-15 cells, but BAY87-2243 was able to inhibit the upregulation of PRV.

[0082] Figure 4A This is a graph showing the results of BAY87-2243 inhibiting the gene expression level of MCT4 by PRV;

[0083] Figure 4B This is a graph showing the results of BAY87-2243 inhibiting the gene expression level of HK2 induced by PRV.

[0084] Figure 4A and Figure 4B ns: no significant difference, *: P <0.05, significant difference; **: P <0.01, the difference is extremely significant; ***: P <0.001, the difference is extremely significant.

[0085] 2.3 Inhibitory effect of BAY87-2243 on PRV proliferation

[0086] 2.3.1 Detection of the intracellular inhibitory effect of BAY87-2243 on PRV

[0087] a. PK-15 cells were cultured at 1×10 4 / well of a 96-well plate, and cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 until the cell density reached 80%, then BAY87-2243 (200nM) diluted in DMEM was added, and a control group of BAY87-2243 (0nM) was set up. After incubation for 1 hour, the culture medium was aspirated and discarded, and the cells were rinsed 3 times with PBS, and the cells were infected with the PRV GD-WH strain at a multiplicity of infection of 1 (MOI=1), and a drug-treated control group was set up.

[0088] b. After incubation at 37°C for 1 hour, discard the virus solution, rinse with PBS three times, and then add DMEM cell maintenance medium containing the original concentration of BAY87-2243 and continue culturing for 12 hours.

[0089] c. Discard the culture medium, rinse with PBS three times, and extract cell DNA using Tissue DNA Kit D3396.

[0090] d. The intracellular viral gene content of PK-15 was detected by SYBR Green fluorescence quantitative PCR. The viral gene and primer sequences are shown in Table 6.

[0091] Table 6 PRV gE gene and its primer sequences

[0092]

[0093] e. The experimental results were statistically analyzed using GraphPad Prism 8 software.

[0094] By measuring the intracellular viral load (gE), refer to Figure 5 The results showed that the intracellular viral load of BAY87-2243-treated cells was significantly lower than that of the virus control group, and BAY87-2243 treatment reduced the TCID of PRV. 50 .

[0095] Figure 5 ns: no significant difference, *: P <0.05, significant difference; **: P <0.01, the difference is extremely significant; ***: P <0.001, the difference is extremely significant.

[0096] 2.3.2 Detection of BAY87-2243 against PRV TCID 50 Impact

[0097] a. PK-15 cells were cultured at 1×10 4 / well of a 96-well plate, and cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 until the cell density reached 80%. Then, BAY87-2243 (200nM) diluted in DMEM was added, and a control group of BAY87-2243 (0nM) was set up. After incubation for 1 hour, the culture medium was discarded, and the cells were rinsed 3 times with PBS. The cells were infected with PRV GD-WH strain at a multiplicity of infection of 1 (MOI=1), and a drug-treated control group was set up.

[0098] b. After incubation at 37°C for 1 hour, discard the virus solution, rinse with PBS three times, and then add DMEM cell maintenance medium containing the original concentration of BAY87-2243 and continue culturing for 12 hours.

[0099] c. Collect the supernatant culture medium and store it in a -80℃ refrigerator for future use.

[0100] d. PK-15 cells were cultured at 1×10 4 100 μL of the supernatant collected in the previous step was diluted 10-fold with DMEM and inoculated into PK-15 cells in a 96-well cell culture plate. Each dilution was repeated 4 times, and a normal cell control group without sample was set up and cultured in a cell culture incubator. The cell growth status was observed every 24 hours, and the number of cytopathic wells was recorded. The observation lasted for 3 to 5 days until the number of cytopathic wells no longer increased.

[0101] e. Calculate the TCID50 of the virus using the Reed-Muench method.

[0102] Through TCID 50 The results of the determination are as follows Figure 6 .

[0103] The results showed that the intracellular viral load of BAY87-2243-treated cells was significantly lower than that of the virus control group, and BAY87-2243-treated cells reduced the TCID of PRV. 50 .

[0104] Figure 5 ns: no significant difference, *: P <0.05, significant difference; **: P <0.01, the difference is extremely significant; ***: P <0.001, the difference is extremely significant.

[0105] Part III Results Analysis:

[0106] 1. PRV infection upregulated the mRNA levels of HK2, MCT, LDHA, and PDK1 in PK-15 cells;

[0107] 2. PRV infection upregulates the protein level of HIF-1α, and BAY87-2243 can inhibit the activation of HIF-1α by PRV infection;

[0108] 3.PRV infection up-regulated the mRNA levels of MCT1 and HK2 in PK-15 cells, but BAY87-2243 could inhibit the up-regulation of PRV;

[0109] Based on the above findings, BAY87-2243 was further used to treat the virus and it was found that it could significantly reduce the TCID of PRV. 50 The above experimental results can prove that BAY87-2243 can be used as a raw material for pseudorabies virus inhibitory drugs.

Claims

1. Use of BAY87-2243 in the preparation of a drug for inhibiting the proliferation of pseudorabies virus.

Images