Application of L-type calcium channel inhibitor in resisting porcine intestinal coronavirus infection

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an L-type calcium channel inhibitor in resisting porcine intestinal coronavirus infection. The porcine intestinal coronavirus comprises any one or more of transmissible gastroenteritis virus, porcine epidemic diarrhea virus and porcine D-type coronavirus. The L-type calcium channel inhibitor is prepared from any one or more of verapamil, nicardipine and nimodipine. Research finds that the L-type calcium channel inhibitor such as verapamil can significantly inhibit porcine intestinal coronavirus infection and mainly plays a role within one hour when porcine intestinal coronavirus infection occurs, and it is indicated that the L-type calcium channel inhibitor mainly hinders the invasion process of viruses. The invention develops a brand new field for application of L-type calcium channel inhibitors such as verapamil, and also provides a new direction for research and development of novel anti-porcine intestinal coronavirus drugs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an application of an L-type calcium ion channel inhibitor in resisting porcine intestinal coronavirus infection. Background Art

[0002] Swine enteric coronaviruses (SECs) are a class of viruses that mainly infect the villus enterocytes of the small intestine of pigs, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine delta coronavirus (PDCoV), etc. SECs have similar genome structures and can cause indistinguishable clinical symptoms such as diarrhea, vomiting, loss of appetite and dehydration in pigs. Especially in suckling piglets, TGEV and PEDV can cause up to 100% mortality, causing huge economic losses to the pig industry.

[0003] The epidemic characteristics of SECs are characterized by the coexistence and genetic diversity of multiple virus strains, which poses a challenge to the protection of existing vaccines. Although a variety of vaccines have been developed for TGEV and PEDV viruses, there is no commercial vaccine available for PDCoV. Factors such as insufficient vaccine protection, the presence of immunosuppressive diseases, and antigenic changes in emerging variants have led to the continued prevalence of SECs in pig herds. At present, there is no effective broad-spectrum antiviral drug for porcine enteric coronavirus on the market. The development of a broad-spectrum antiviral drug that can simultaneously inhibit PEDV, TGEV, and PDCoV for the prevention and treatment of porcine viral diarrhea is of great significance to the pig industry.

[0004] Voltage-gated calcium channels (VGCC or Ca v ) is a type of transmembrane protein that controls the flow of calcium ions into and out of cells. It plays an important role in physiological processes such as gene expression, neurotransmitter release, and muscle contraction. According to the gating characteristics of calcium ion currents, Ca v It can be divided into L-type, P / Q-type, N-type, R-type and T-type. Currently, there are many drugs on the market for L-type calcium ion channels, mainly divided into dihydropyridines (nifedipine, amlodipine and lercanidipine), phenylalkyls (verapamil) and diazepam. These drugs mainly play a role in the treatment of mental illness or arrhythmia. It is reported that calcium ions or voltage-gated calcium ion channels play an important role in mediating the invasion of various viruses, but there is no existing technology to use them for the prevention and treatment of porcine enteric coronavirus infection.

[0005] Some existing technologies have studied drugs for treating porcine enteric coronavirus infections. For example, the invention patent with publication number CN117562887B discloses the use of 4-octyl itaconic acid in the preparation of drugs and / or feed additives for treating porcine enteric coronavirus infections. The invention study found that 4-octyl itaconic acid has a significant inhibitory effect on porcine enteric coronavirus during the replication stage. When the concentration is between 0.25mM and 0.4mM, it does not show cytotoxicity, and the antiviral effect is proportional to the concentration. For another example, the invention patent with publication number CN113018296B discloses the use of melatonin in anti-swine coronavirus, and its mechanism of action is that melatonin inhibits the invasion process of the virus. Its research found that melatonin has the effect of inhibiting TGEV, PEDV and PDCoV at the intracellular level. Summary of the invention

[0006] In view of this, in order to overcome the shortcomings of existing porcine coronavirus prevention and treatment technologies, the present invention has conducted research on drugs against porcine enteric coronavirus. The present invention has found that L-type calcium channel inhibitors such as verapamil have broad-spectrum antiviral activity against porcine enteric coronaviruses such as TGEV, PEDV and PDCoV; and the drug mainly hinders the invasion stage of the virus, which indicates that L-type calcium channel inhibitors such as verapamil have the potential to be developed as drugs for the treatment of porcine enteric coronavirus infection.

[0007] One of the purposes of the present invention is to provide an application of an L-type calcium channel inhibitor in the preparation of a product for preventing and / or treating porcine enteric coronavirus infection.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] Application of L-type calcium ion channel inhibitors in the preparation of products for preventing and / or treating porcine enteric coronavirus infection.

[0010] Furthermore, the porcine enteric coronavirus includes any one or more of transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine delta coronavirus.

[0011] Furthermore, the L-type calcium channel inhibitor includes any one or more of verapamil, nicardipine, and nimodipine.

[0012] Preferably, the L-type calcium channel inhibitor is verapamil.

[0013] Furthermore, the product includes any one or more of medicines, feed additives, and vaccines.

[0014] Furthermore, the final concentration of the L-type calcium channel inhibitor is 0.1 to 1000 μM.

[0015] Preferably, the final concentration of the verapamil is 1 to 100 μM, and / or the final concentration of the nicardipine is 1 to 200 μM, and / or the final concentration of the nimodipine is 1 to 200 μM.

[0016] Preferably, the final concentration of the verapamil is 1 to 50 μM, and / or the final concentration of the nicardipine is 1 to 100 μM, and / or the final concentration of the nimodipine is 1 to 100 μM.

[0017] More preferably, the final concentration of the verapamil is 25 to 50 μM, and / or the final concentration of the nicardipine is 25 to 100 μM, and / or the final concentration of the nimodipine is 25 to 100 μM.

[0018] Furthermore, the L-type calcium channel inhibitor inhibits viral infection or cytopathic effect in a dose-dependent manner.

[0019] Preferably, the cells include any one or more of green monkey kidney cells, pig testicular cells, and pig jejunal epithelial primary cells.

[0020] The second object of the present invention is to provide an application of an L-type calcium ion channel inhibitor in the preparation of a product for inhibiting early viral invasion of porcine intestinal coronavirus infection.

[0021] To achieve the above object, the present invention adopts the following technical solutions:

[0022] The use of L-type calcium ion channel inhibitors in the preparation of products for inhibiting early viral invasion of porcine enteric coronavirus infection, wherein the porcine enteric coronavirus includes any one or more of transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine delta coronavirus; the L-type calcium ion channel inhibitor includes any one or more of verapamil, nicardipine, and nimodipine; and the product includes any one or more of drugs, feed additives, and vaccines.

[0023] Preferably, the L-type calcium channel inhibitor is verapamil.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides for the first time an application of an L-type calcium channel inhibitor in the prevention of porcine intestinal coronavirus infection, opening up a new field for L-type calcium channel inhibitors such as verapamil in the prevention and treatment of porcine intestinal coronavirus. In the early stage of the present invention, several porcine intestinal coronavirus susceptible cells (Vero, ST and porcine jejunal epithelial primary cells) were treated with L-type calcium channel inhibitors and it was found that the infection ability of porcine intestinal coronaviruses such as PEDV, TGEV, and PDCoV was significantly inhibited. Further studies have found that Verapamil mainly works within 1 hour of porcine intestinal coronavirus infection, indicating that Verapamil mainly hinders the invasion process of the virus. In order to simulate the inhibitory effect of Verapamil on porcine intestinal coronavirus infection in vivo, the present invention verified the antiviral activity of Verapamil in primary jejunal epithelial cells isolated from pigs, and found that Verapamil can also significantly hinder porcine intestinal coronavirus infection. The research results show that in addition to playing an important role in the treatment of hypertension and arrhythmia, Verapamil also has the potential to be developed as an antiviral drug for inhibiting porcine intestinal coronaviruses such as PEDV, TGEV and PDCoV. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of the effects of Vero cells treated with Verapamil, Nifedipine, Nicardipine and Nimodipine on PEDV infection are shown in the figure. Figure 1 -A is the result of the effect of PEDV infection on ST cells after treatment with Nifedipine;

[0027] Figure 1 -B is the result of the effect of PEDV infection on Vero cells treated with Nicardipine; Figure 1 -C is the result of the effect of PEDV infection on Vero cells treated with Verapamil; Figure 1 -D is the result of the effect of PEDV infection on Vero cells treated with Nimodipine;

[0028] Figure 2 This is a graph showing the cell viability of Vero cells after treatment with Verapamil, Nifedipine, Nicardipine and Nimodipine, where: Figure 2 -A is a graph showing the cell viability of Vero cells after treatment with Nifedipine; Figure 2 -B is a graph showing the cell viability of Vero cells after treatment with Nicardipine; Figure 2 -C is a graph showing the cell viability of Vero cells after treatment with Verapamil; Figure 2-D is a graph showing the cell viability of Vero cells after treatment with Nimodipine;

[0029] Figure 3 The figure shows the cytopathic effect of PEDV infection in Vero cells after treatment with Verapamil, Nifedipine, Nicardipine and Nimodipine;

[0030] Figure 4 The results of the effects of TGEV infection on ST cells after treatment with Verapamil, Nifedipine, Nicardipine and Nimodipine are shown in the figure. Figure 4 -A is the result diagram of the effect of TGEV infection on ST cells after treatment with Nifedipine; Figure 4 -B is the result of the effect of TGEV infection on ST cells after treatment with Nicardipine; Figure 4 -C is the result diagram of the effect of TGEV infection on ST cells after treatment with Verapamil; Figure 4 -D is the result of the effect of TGEV infection on ST cells after treatment with Nimodipine;

[0031] Figure 5 Figure 2 is a graph showing the cell viability of ST cells after treatment with Verapamil, Nifedipine, Nicardipine and Nimodipine, where: Figure 5 -A is a graph showing the cell viability of ST cells after treatment with Nifedipine; Figure 5 -B is a graph showing the cell viability of ST cells after treatment with Nicardipine; Figure 5 -C is a graph showing the cell viability of ST cells after treatment with Verapamil; Figure 5 -D is a graph showing the cell viability of ST cells after treatment with Nimodipine;

[0032] Figure 6 The figure shows the cytopathic effect of TGEV infection in ST cells after treatment with Verapamil, Nifedipine, Nicardipine and Nimodipine;

[0033] Figure 7 This is an indirect immunofluorescence image of TGEV infection in ST cells treated with Verapamil;

[0034] Figure 8 The results of the effect of Verapamil treatment on ST cells infected with TGEV are shown in Figure 2. Figure 8 -A is the titration graph of PDCoV infection virus after ST cells were treated with Verapamil; Figure 8 -B is a diagram showing the cytopathic effect of PDCoV infection in ST cells after treatment with Verapamil;

[0035] Fig. 9 This is the test result of Verapamile on the early effects of porcine enteric coronavirus infection, among which: Fig. 9 -A is the test result of the effect of Verapamile on the early stage of PEDV infection. Fig. 9 -B is the test result of the effect of Verapamile on the early stage of TGEV infection. Fig. 9 -C is a graph showing the test results of the effect of Verapamile on the early stage of PDCoV infection;

[0036] Fig.10 This is the result of Verapamil treatment of porcine jejunal epithelial primary cells on porcine enteric coronavirus infection, among which, Fig.10 -A is the result of the treatment of porcine jejunal epithelial primary cells with Verapamil on PEDV infection. Fig.10 -B is the result of Verapamil treatment of porcine jejunal epithelial primary cells on TGEV infection. Fig.10 -C is the result of the Verapamil treatment of porcine jejunal epithelial primary cells on PDCoV infection. Fig.10 -D is the result of viability assay of porcine jejunal epithelial primary cells treated with Verapamil. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0038] In the embodiment of the present invention, Verapamil is a derivative of papaverine and a calcium channel blocker, and its molecular formula is C 27 H 38N2O4, chemical name: 5-((3,4-dimethoxyphenethyl)methylamino)-2-(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile. Verapamil is widely used in clinical practice. The drug was first used as a coronary vasodilator in 1962. In recent years, it has been used to treat hypertension, angina pectoris, arrhythmia, cerebrovascular disease, finger vasospasm, abdominal pain, esophageal achalasia, migraine, pulmonary hypertension and prevent premature birth. Verapamil mainly inhibits calcium ion influx and reduces the rate of automatic depolarization during diastole, thereby slowing down the impulse and conduction velocity of the sinus node, which helps to eliminate atrioventricular node reentry. In addition, verapamil can dilate peripheral blood vessels, reduce blood pressure, increase coronary flow, and improve myocardial oxygen supply.

[0039] In the embodiments of the present invention, the porcine enteric coronaviruses such as PEDV, TGEV and PDCoV involved in the experiment were isolated by researchers in this laboratory. The PEDV virus was amplified in green monkey kidney cells (Vero) and stored at -70°C, and TGEV and PDCoV were amplified in pig testicular cells (ST) and stored at -70°C.

[0040] In the examples of the present invention, Vero cells and ST cells were provided by our laboratory, and primary porcine jejunal epithelial cells were provided by the Institute of Bioengineering, Chongqing Academy of Animal Husbandry Sciences.

[0041] In the examples of the present invention, Verapamil was purchased from MedChemExpress.

[0042] Example 1. Verapamile inhibits porcine enteric coronavirus infection

[0043] (1) Infectious titers or cytopathic effects of three viruses treated with Verapamil

[0044] Vero cells, porcine testicular cells or porcine jejunal epithelial primary cells were pretreated with different concentrations of L-type calcium channel inhibitors: Verapamil (0, 1, 5, 10, 25, 50 μM), nifedipine (0, 10, 50, 100, 300, 600 μM), nicardipine (0, 1, 10, 25, 50, 100 μM) and nimodipine (0, 1, 10, 25, 50, 100 μM) for 1 h, and then infected with PEDV, TGEV or PDCoV at an MOI of 0.1. After 48 h of infection, the cytopathic effect was observed and the cell supernatant was collected. The TCID 50 Methods Virus titer was determined, cells were fixed with 4% paraformaldehyde, and cytopathic effect was observed.

[0045] (2) Cell activity assay

[0046] After the cells were treated with different concentrations of Verapamil, Nifedipine, Nicardipine and Nimodipine, the culture supernatant was discarded, and Cell Titer-Glo 2.0Assay reagent was added. The cells were incubated in the dark for 5 minutes, and the ATP content of the samples was detected by a multifunctional microplate reader to calculate the cell viability.

[0047] (3) Indirect immunofluorescence observation

[0048] After pretreatment of pig testicular cells (ST) with different concentrations of Verapamil for 1 hour, the cells were infected with TGEV at MOI = 0.1. After 24 hours of infection, the cell supernatant was discarded, the cells were fixed with 4% paraformaldehyde, and the membrane was permeabilized with 0.1% Triton. Mouse anti-TGEV specific antibodies and goat anti-mouse FITC fluorescent secondary antibodies were incubated to observe the infection rate of ST cells.

[0049] The results are as follows Figures 1 to 8 As shown, except for Nifedipine treatment, there was no significant difference. The infection titer, infection rate or cytopathic effect of the three viruses were significantly inhibited after Verapamil, Nicardipine and Nimodipine treatment, and the inhibition effect was better with the increase of drug concentration; at the same time, Verapamil, Nicardipine and Nimodipine at the above concentrations were not toxic to Vero or ST.

[0050] Example 2. Inhibition of early stage of porcine enteric coronavirus infection

[0051] To study the early effects of Verapamil on enteric coronavirus infection, Vero or ST cells treated with Verapamil (10 μM) for different times (-1, 0, 1, 3, and 5 h) were infected with PEDV, TGEV, or PDCoV (MOI = 0.1). The cells were collected 8 h after infection, and the viral RNA levels in the infected cells were detected by qPCR.

[0052] The results are as follows Fig. 9 As shown, the inhibitor mainly exerts its effect within 1 hour after porcine enteric coronavirus infection, indicating that Verapamil mainly hinders the invasion of porcine enteric coronavirus.

[0053] Example 3. Verapamil treatment of porcine jejunal epithelial primary cells inhibits porcine enteric coronavirus infection

[0054] PEDV, TGEV, and PDCoV mainly infect pig jejunal epithelial cells. To further study whether Verapamil inhibits the infection of pig enteric coronavirus in pig jejunal epithelial cells, we treated the isolated jejunal epithelial primary cells with different concentrations of Verapamil (0, 1, 5, 10, 25 μM), and then infected them with PEDV, TGEV, and PDCoV.

[0055] The results are as follows Fig.10 As shown, the infection titer of porcine enteric coronavirus was significantly reduced after Verapamil was treated with porcine jejunal primary epithelial cells, indicating that Verapamil has the potential to be developed as an anti-porcine enteric coronavirus drug.

Claims

1. Application of L-type calcium channel inhibitors in the preparation of products for preventing and / or treating porcine enteric coronavirus infection.

2. The use according to claim 1, characterized in that: The porcine enteric coronavirus includes any one or more of transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine delta coronavirus.

3. The use according to claim 1, characterized in that: The L-type calcium channel inhibitor includes any one or more of verapamil, nicardipine, and nimodipine.

4. The use according to claim 1, characterized in that: The product includes any one or more of medicines, feed additives, and vaccines.

5. The use according to claim 1, characterized in that: The final concentration of the L-type calcium channel inhibitor is 0.1-1000 μM.

6. The use according to claim 5, characterized in that: The final concentration of the verapamil is 1-100 μM, and / or the final concentration of the nicardipine is 1-200 μM, and / or the final concentration of the nimodipine is 1-200 μM.

7. The use according to claim 1, characterized in that: The L-type calcium channel inhibitor inhibits viral infection or cytopathic effect in a dose-dependent manner.

8. The use according to claim 7, characterized in that: The cells include any one or more of green monkey kidney cells, pig testicular cells, and pig jejunal epithelial primary cells.

9. Use of an L-type calcium channel inhibitor in the preparation of a product for inhibiting early viral invasion of porcine intestinal coronavirus infection, characterized in that: The porcine enteric coronavirus includes any one or more of transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine delta coronavirus; the L-type calcium channel inhibitor includes any one or more of verapamil, nicardipine, and nimodipine; and the product includes any one or more of drugs, feed additives, and vaccines.

10. The use according to claim 9, characterized in that: The L-type calcium channel inhibitor is verapamil.

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