AU2020103362A4

AU2020103362A4 - Application of EGCG in the preparation for the prevention and/or treatment of the PRV infection, preparation for the prevention and/or treatment of PRV infection.

Info

Publication number: AU2020103362A4
Application number: AU2020103362A
Authority: AU
Inventors: Song Gao; Tingting GUO; Changchao Huan; Jinbiao Liu; Weiyin XU
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2020-11-10
Filing date: 2020-11-10
Publication date: 2021-01-21
Anticipated expiration: 2028-11-10

Abstract

The invention provides the application of epigallocatechin gallate in the preparation for the prevention and/or treatment of the infection of porcine pseudorabies virus, and the preparation for the prevention and/or treatment of the porcine pseudorabies virus, belonging to the technical field of treatment of porcine pseudorabies virus infection. The concentration of the epigallocatechin gallate in the medicine is 10-100 mol/L. In the invention, the anti infection effect of the epigallocatechin gallate against the porcine pseudorabies virus is very remarkable: The epigallocatechin gallate can reduce the cytopathy caused by the porcine pseudorabies virus, inhibit the adsorption, endocytosis and replication of the porcine pseudorabies virus, and may directly kill the porcine pseudorabies virus. More importantly, the epigallocatechin gallate can prevent and/or treat the mice infected with porcine pseudorabies virus, which provides the scientific and reliable theoretical basis for the clinical treatment of porcine pseudorabies. -1/8 Mock 0 10 20 50 100 PM 12h 24h Figure 1

Description

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Figure 1

Application of EGCG in the preparation for the prevention and/or treatment of the PRV infection, preparation for the prevention and/or treatment of PRV infection.

TECHNICAL FIELD

The invention belongs to the technical field of treatment for porcine pseudorabies virus infection, in particular to the application of epigallocatechin gallate in the preparation for the prevention and/or treatment of porcine pseudorabies virus infection, and prevention and/or treatment of porcine pseudorabies virus infection.

BACKGROUND

The porcine pseudorabies virus is the linear double-stranded DNA molecule, belonging to Herpesviridae, porcine herpesvirus genus. The virus particles are coated with the membrane and have strong resistance to the external environment. Pseudorabies virus is pantropic and can proliferate in a variety of tissue culture cells, among which the rabbit kidney and pig kidney cells (including 1st generation cell and passage cell lines) are the most sensitive and cause obvious cytopathy. The pig is the reservoir host of pseudorabies virus, and the infected pig, the viruliferous pig and the viruliferous rat are the important sources of infection of the disease. In the swine herd, the virus is spread mainly through nasal secretion. In addition, latex and semen are also possible ways of transmission. After the occurrence of pseudorabies in pigs, the clinical symptoms vary with the age of the day. The fatality rate of piglets under the age of 15 days is 100%; The pregnant sows would be infertile and miscarried; The male pigs show testicular swelling, atrophy, loss of breeding function and so on. It may cause huge economic losses to the pig-raising industry.

Pseudorabies (PR), also known as AujeszKy's disease, is an acute viral infection disease characterized by fever, severe itching (except in pigs), respiratory and nervous system diseases, and encephalomyelitis, co-infected among a variety of livestock and wild animals caused by the Suid herpesvirus 1 (SuHV-1) or known as Pseudorabies virus (PRV). The disease is an explosive epidemic in pigs. It can cause miscarriage and stillbirth of pregnant sows, sterility of boars, mass death of newborn piglets, dyspnea and stagnation of growth of fattening pigs, etc. It is a major infectious disease that endangers the global pig-raising industry. Since 2011, in many areas of our country, there has been serious outbreak of porcine pseudorabies, and these pig farms are inoculated with Bartha-K61 vaccine. The newly emerged pseudorabies virus variant is different from the classic PRV strain, it has high pathogenicity to pigs of all ages, and shows different clinical symptoms, which means: that the existing vaccine may not be able to deal with the current PRV epidemic strain, and can not provide complete immune protection. In short, pig farming faces huge risks from PRV. More importantly, the host spectrum of pseudorabies virus is very wide. In addition to pigs, cattle, sheep, dogs, cats and other vertebrates, there are reports showing that mutated porcine pseudorabies virus can also infect people. Therefore, the prevention and control of PRV infection is particularly urgent and important.

At present, there is no effective treatment for mutated PRV in China, which can only be controlled by early prevention and strengthening of health management. In order to reduce the economic loss of pig-raising industry, it is urgent to develop effective medicines to prevent and treat mutated PRV.

SUMMARY

In view of this, the present invention aims to provide the application of epigallocatechin gallate in the preparation for the prevention and/or treatment of the infection of porcine pseudorabies virus. The preparation has low cost, no side effect and remarkable curative effect.

To realize the above purpose, the invention provides the following technical scheme:

The invention provides the application of epigallocatechin gallate in the preparation for the prevention and/or treatment of the infection of porcine pseudorabies virus.

Preferably, the epigallocatechin gallate prevents and/or treats the porcine pseudorabies by inhibiting the adsorption, endocytosis and replication of porcine pseudorabies virus.

Preferably, the epigallocatechin gallate prevents and/or treats the porcine pseudorabies by killing the porcine pseudorabies virus directly.

The invention provides a preparation for the prevention and/or treatment of the porcine pseudorabies virus infection. The concentration of epigallocatechin gallate in the preparation is 10~100 mol/L.

Preferably, the concentration of epigallocatechin gallate in the preparation is -80 [mol/L.

Preferably, the concentration of epigallocatechin gallate in the preparation is ~60 mol/L.

Preferably, the solvent of the preparation is PBS buffer solution or DMSO.

Preferably, the concentration of the PBS buffer solution is 0.05-0.15 mol/L.

Preferably, the concentration of the PBS buffer solution is 0.08-0.12 mol/L.

Preferably, the preparation is obtained by dissolving the epigallocatechin gallate in the solvent.

The beneficial effects of the invention are as follows: The invention provides the application of epigallocatechin gallate in the preparation for the prevention and/or treatment of porcine pseudorabies virus infection; The anti-infection effect of epigallocatechin gallate against porcine pseudorabies virus infection is very remarkable: The epigallocatechin gallate can reduce the cytopathy caused by porcine pseudorabies virus, inhibit the adsorption, endocytosis and replication of porcine pseudorabies virus, and can directly kill the porcine pseudorabies virus, providing the scientific and reliable theoretical basis for the clinical treatment of porcine pseudorabies. At the same time, epigallocatechin gallate is the most abundant bioactive polyphenol found in the solid green tea extract, which has the advantages of wide source of medicine, easy availability of raw materials and low cost; The epigallocatechin gallate is from traditional Chinese medicine, which is metabolized by animal body and has little environmental pollution and no toxic side effects on animals; The epigallocatechin gallate has great clinical biological significance with anti-oxidant, anti-atherosclerosis, anti-thrombosis, anti-vascular proliferation, anti neoplastic and other pharmacological effects.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 The effect of EGCG on the cytopathy of PK-15B6 cells infected by porcine pseudorabies virus infection is observed under microscope.

Figure 2 Effect of EGCG on porcine pseudorabies virus infection in PK-15B6 cells; where, A for the effect measured by Western blot of EGCG on porcine pseudorabies virus infection in PK-15B6 cells; B for the effect measured by TCID50 of EGCG on porcine pseudorabies virus infection in PK-15B6 cells; C for the effect measured by fluorescent quantitation PCR of EGCG on porcine pseudorabies virus infection in PK-15B6 cells;

Figure 3 shows the effect of EGCG on the adsorption and endocytosis of porcine pseudorabies virus in PK-15B6 cells, where A for the effect measured by Western blot of EGCG on adsorption and endocytosis of porcine pseudorabies virus in PK B6 cells; B for the effect measured by TCID50 of EGCG on adsorption and endocytosis of porcine pseudorabies virus in PK-15B6 cells; C for the effect measured by fluorescent quantitation PCR of EGCG on adsorption and endocytosis of porcine pseudorabies virus in PK-15B6 cells;

Figure 4 shows the effect of EGCG on the adsorption of porcine pseudorabies virus in PK-15B6 cells, where A for the effect measured by Western blot of EGCG on adsorption of porcine pseudorabies virus in PK-15B6 cells; B for the effect measured by TCID50 of EGCG on adsorption of porcine pseudorabies virus in PK-15B6 cells; C for the effect measured by fluorescent quantitation PCR of EGCG on adsorption of porcine pseudorabies virus in PK-15B6 cells;

Figure 5 shows the effect of EGCG on the endocytosis of porcine pseudorabies virus in PK-15B6 cells, where A for the effect measured by Western blot of EGCG on endocytosis of porcine pseudorabies virus in PK-15B6 cells; B for the effect measured by fluorescent quantitation PCR of EGCG on endocytosis of porcine pseudorabies virus in PK-15B6 cells; C for the effect measured by TCID50 of EGCG on endocytosis of porcine pseudorabies virus in PK-15B6 cells;

Figure 6 shows the effect measured by Western blot of EGCG on the replication of porcine pseudorabies virus in PK-15B6 cells;

Figure 7 shows the direct killing effect of EGCG on porcine pseudorabies virus; where A for the direct killing effect measured by Western blot of EGCG on porcine pseudorabies virus. B for the direct killing effect measured by TCID50 of EGCG on porcine pseudorabies virus;

Figure 8 shows the verification the protective rate of EGCG against infected mice through the survival rate of mice.

DESCRIPTION OF THE INVENTION

The invention provides the application of epigallocatechin gallate in the preparation for the prevention and/or treatment of porcine pseudorabies virus infection. In the invention, the anti-infection effect of epigallocatechin gallate against porcine pseudorabies virus infection is very remarkable: The epigallocatechin gallate can reduce the cytopathy caused by porcine pseudorabies virus, inhibit the adsorption, endocytosis and replication of porcine pseudorabies virus, and can directly kill the porcine pseudorabies virus; Through the in vivo experiments of rats, the epigallocatechin gallate can prevent and/or treat the mice infected with porcine pseudorabies virus. The invention has no special requirements for the source of the epigallocatechin gallate, and it can be prepared by using the conventional commercial product in this field or by self-preparation.

The invention also provides a preparation for the prevention and/or treatment of the porcine pseudorabies virus infection. The concentration of epigallocatechin gallate in the preparation was 10~100 mmol/L, preferably 20-80 mmol/L, more preferably -60 mmol/L, most preferably 50 mmol/L. In the invention, the medicine was preferably the liquid preparation, and the solvent of the medicine was preferably the PBS buffer solution or DMSO; When the solvent was the PBS buffer solution, the concentration of the PBS buffer solution was preferably 0.05-0.15 mol/L, more preferably 0.08-0.12 mol/L, most preferably 0.1 mol/L.

In the present invention, the medicine was preferably obtained by dissolving the epigallocatechin gallate in the solvent. The invention has no special limitation on the method and time of dissolution, while complete dissolution would be sufficient.

The following embodiments provide a detailed description of the technical scheme of the present invention, but they cannot be construed as limiting the scope of protection of the present invention.

EMBODIMENT 1

A preparation for the prevention and/or treatment of porcine pseudorabies virus infection, which was obtained by dissolving epigallocatechin gallate in 0.1 mol/L of PBS buffer solution. The concentration of epigallocatechin gallate was 50 mmol/L.

EMBODIMENT 2

A preparation for the prevention and/or treatment of porcine pseudorabies virus infection, which was obtained by dissolving epigallocatechin gallate in 0.15 mol/L of PBS buffer solution. The concentration of epigallocatechin gallate was 40 mmol/L.

EMBODIMENT 3

A preparation for the prevention and/or treatment of porcine pseudorabies virus infection, which was obtained by dissolving epigallocatechin gallate in 0.1 mol/L of PBS buffer solution. The concentration of epigallocatechin gallate was 100 mmol/L.

EMBODIMENT 4

A preparation for the prevention and/or treatment of porcine pseudorabies virus infection, which was obtained by dissolving epigallocatechin gallate in DMSO. The concentration of epigallocatechin gallate was 60 mmol/L.

EMBODIMENT 5

Effect of epigallocatechin gallate on cytopathy induced by porcine pseudorabies virus

The PK-15B6 cells (purchased from China Institute of Veterinary Drug Control) were diluted with nutrient solution containing 4% of fetal bovine serum and counted. The cells with concentration of 5x10 5/pore were placed in the six-hole plate and placed in the incubator at 37°C containing 5%of C02. After the cells grew to 70~80% of density (about 16 h), the cells were washed twice with PBS solution. The cells were pretreated with PBS, EGCG at different concentrations (10 M, 20 M, 50 M, 100

[tM) for 1 hour separately, and then infected with PRV XJ5 strain (0.1 MOI) of porcine pseudorabies virus. After 1 hour, the medium was changed. The cells were infected for 24 hours along with the medicine. Under the microscope, the morphological changes and cytopathy situations of the cells were observed. According to the results of Figure 1, EGCG could reduce the cytopathy caused by porcine pseudorabies virus, indicating that EGCG may reduce the infection of porcine pseudorabies virus.

EMBODIMENT 6

The inhibitory effect of epigallocatechin gallate on porcine pseudorabies virus infection in PK-15B6 cells (Western blot, fluorescence quantitative PCR and TCID50 experiment)

(1) The inhibitory effect measured by Western blot of epigallocatechin gallate on porcine pseudorabies virus infection in PK-15B6 cells

PK-15B6 cell digestion was diluted with DMEM nutrient solution containing FBS with volume concentration of 4%, and was dropwise added to the 6-hole plate at the concentration of 5x10 5/pore, placed in the incubator at 37 °C and 5%of C02until the cells attached the wall to form the single layer (about 17-18 h), and then was washed with the PBS solution for three times. After the residual FBS was completely absorbed, 1mL of EGCG diluted by DMEM without serum to corresponding concentration (10 M, 20 M, 50 M, 100 M) was added. After incubation with PK B6 cells at 37 °C for 1 h, the cells were infected with porcine pseudorabies virus with the presence of corresponding concentration of EGCG (10 M, 20 M, 50 M, 100 jM). After incubation in the incubator at 37 °C and 5% CO2 for 1 h,itwas replaced with 2mL of DMEM nutrient solution containing 2% of FBS with the presence of corresponding concentration of EGCG (10 M, 20 M, 50 M, 100 M). Then it was incubated in the incubator at 37 °C and 5% C02 for 24 h, and then the supernatant cells were collected (1 mL at -70 °C for later TCID50 experiment). After washing the residue with PBS for 3 times, adding 2xprotein loading to collect the cell sample. It was cooked at 96 °C in the metal bath for 15 min, then the Western blot test was carried out. It was found that EGCG decreased the expression of gE and UL42 protein of porcine pseudorabies virus (A in Figure 2). It was tentatively proved that EGCG decreased the infection of porcine pseudorabies virus.

(2) The inhibitory activity measured by TCID50 of epigallocatechin gallate on PK-15B6 cells against porcine pseudorabies virus infection.

Vero cell digestion was diluted with DMEM nutrient solution of 8% FBS in volume concentration, and was dropwise added to the 96-hole plate at the concentration of 2x10/pore, placed in the incubator at 37 °C and 5% of C02 until the cells attached the wall to form the single layer, and then was washed with PBS for three times. After the residual solution was completely absorbed, the virus solution of different concentration diluted by DMEM without serum was added, each concentration repeated for 8 times. 1.5 h after the infection, it was replaced with the DMEM with 2% of FBS for maintaining, and the cytopathy situation was observed 72 h after the infection. It was found that EGCG decreased the viral titer of porcine pseudorabies virus infection (B in Figure 2), which confirmed that EGCG decreased the infection of porcine pseudorabies virus.

(3) The inhibitory activity measured by fluorescence quantitative PCR of epigallocatechin gallate on PK-15B6 cells against porcine pseudorabies virus infection.

PK-15B6 cell digestion was diluted with DMEM nutrient solution containing FBS with volume concentration of 4%, and was dropwise added to the 6-hole plate at the concentration of 5x10 5/pore, placed in the incubator at 37 °C and 5% of C02 until the cells attached the wall to form the single layer (about 17-18 h), and then was washed with the PBS solution for three times. After the residual FBS was completely absorbed, 1mL of DMEM diluted by DMEM without serum to corresponding concentration (10 M, 20 M, 50 M, 100 M) was added. After incubation with PK B6 cells at 37 °C for 1 h, the cells were infected with porcine pseudorabies virus with the presence of corresponding concentration of EGCG (10 M, 20 , t50 0 t100 ). After incubation in the incubator at 37 °C and 5% C02 for 24 h, it was washed with PBS for three times, and then 1mL of DMEM nutrient solution without serum was added. It was placed in the refrigerator at -70 °C for repeated freeze thawing for 3 times. Then DNA was extracted for fluorescence quantitative PCR test. The PCR primers used were: gB94F:5'-ACAAGTTCAAGGCCCACATCTAC-3' (SEQ IDNo.1); gB94R:5'-GTCCGTGAAGCGGTTCGTGAT(SEQIDNo.2); gB probe 5' FAM-ACGTCATCGTCACGACC-TAMRA-3'(SEQ IDNo.3). The 20tL of reaction system contained 0.2L of DMSO, 0.5tL of Taq DNA polymerase, and 12.9 L of water without RNase. There were 45 cycles of amplification at 95 °C 600 s, 95 °C 10 s, 62 °C 20 s. As shown in C in Figure 2, EGCG could decrease the number of nucleic acid copies of porcine pseudorabies virus, which confirmed that EGCG could decrease the infection of porcine pseudorabies virus.

EMBODIMENT 7

Effects of epigallocatechin gallate on adsorption and endocytosis of porcine pseudorabies virus (Western blot, fluorescent quantitative PCR and TCID50 test)

(1) Effect of epigallocatechin gallate measured by Western blot on the adsorption and endocytosis of porcine pseudorabies virus in PK-15B6 cells

PK-15B6 cell digestion was diluted with DMEM nutrient solution containing FBS with concentration of 4%, and was dropwise added to the 6-hole plate at the concentration of 5x10 5/pore, placed in the incubator at 37 °C and 5% of C02 until the cells attached the wall to form the single layer (about 17-18 h), and then was washed with the PBS solution for three times. After the residual FBS was completely absorbed, 1mL of EGCG diluted by DMEM without serum to corresponding concentration (10 M, 20 M, 50 M, 100 M) was added. After incubation with PK B6 cells at 37 °C for 1 h, it was replaced with DMEM without serum, and the cells were infected at 4 °C with porcine pseudorabies virus with the presence of corresponding concentration of EGCG (10 M, 20 M, 50 M, 100 M). After incubation for 1 h, it was washed with cold PBS for three times, and was added 1mL of DMEM with 2% of FBS with the presence of corresponding concentration of EGCG (10 M, 20 M, 50 M, 100 jM). After incubation at 37 °C for 1 h, it was washed with citric acid for three times and then washed with PBS solution for three times and the residual solution was completely absorbed. It was replaced with 2mL of DMEM nutrient solution with 2% of FBS for maintaining, incubated in the incubator at 37°C andC02 for 24 h, and then the supernatant cells were collected (1 mL at -70 °C for later TCID50 experiment). 2xprotein loading was added to collect the cell sample. It was cooked at 96 °C in the metal bath for 15 min, then the Western blot test was carried out. The results were shown in A in Figure 3. It was found that EGCG decreased the expression of gE and UL42 protein of porcine pseudorabies virus. It was tentatively proved that EGCG decreased the adsorption and endocytosis of porcine pseudorabies virus.

(2) Effects of epigallocatechin gallate measured by TCID50 on the adsorption of porcine pseudorabies virus in PK-15B6 cells.

Vero cell digestion was diluted with DMEM nutrient solution of 8% FBS in concentration, and was dropwise added to the 96-hole plate at the concentration of 2x10 3/pore, placed in the incubator at 37 °C and 5%of C02until the cells attached the wall to form the single layer, and then was washed with PBS for three times. After the residual solution was completely absorbed, the virus solution of different concentration diluted by DMEM without serum was added, each concentration repeated for 8 times. 1.5 h after the infection, it was replaced with the DMEM with 2% of FBS for maintaining, and the cytopathy situation was observed 72 h after the infection. The results were shown in B in Figure 3. It was found that EGCG decreased the viral titer of porcine pseudorabies virus infection, which confirmed that EGCG decreased the adsorption and endocytosis of porcine pseudorabies virus.

(3) Effects of epigallocatechin gallate measured by fluorescence quantitative PCR on adsorption and endocytosis of porcine pseudorabies virus in PK-15B6 cells.

PK-15B6 cell digestion was diluted with DMEM nutrient solution containing FBS with concentration of 4%, and was dropwise added to the 6-hole plate at the concentration of 5x10 5/pore, placed in the incubator at 37 °C and 5 %of C02until the cells attached the wall to form the single layer (about 17-18 h), and then was washed with the PBS solution for three times. After the residual FBS was completely absorbed, 1mL of EGCG diluted by DMEM without serum to corresponding concentration (10 M, 20 M, 50 M, 100 M) was added. After incubation with PK B6 cells at 37 °C for 1 h, it was replaced with cold DMEM without serum, and the cells were infected at 4 °C with porcine pseudorabies virus with the presence of corresponding concentration of EGCG (10 M, 20 M, 50 M, 100 M). After incubation for 1 h, it was washed with cold PBS for three times, and was added 1mL of DMEM with 2% of FBS with the presence of corresponding concentration of EGCG (10 M, 20 M, 50 M, 100 jM). After incubation at 37 °C for 1 h, it was washed with citric acid for three times and then washed with PBS solution for three times, and the residual solution was completely absorbed. It was replaced with 2mL of DMEM nutrient solution with 2% of FBS for maintaining, added 1mL of DMEM without serum. It was placed in the refrigerator at -70 °C for repeated freeze thawing for 3 times. Then DNA was extracted for fluorescence quantitative PCR test. As shown in C in Figure 3, EGCG could decrease the number of nucleic acid copies of porcine pseudorabies virus, which confirmed that EGCG could decrease the adsorption and endocytosis of porcine pseudorabies virus.

EMBODIMENT 8

Effect of epigallocatechin gallate on adsorption of porcine pseudorabies virus (Western blot, fluorescent quantitative PCR and TCID50 test)

(1) Effect of epigallocatechin gallate measured by Western blot on the adsorption of porcine pseudorabies virus in PK-15B6 cells

PK-15B6 cell digestion was diluted with DMEM nutrient solution containing FBS with concentration of 4%, and was dropwise added to the 6-hole plate at the concentration of 5x10 5/pore, placed in the incubator at 37 °C and 5% of C02 until the cells attached the wall to form the single layer (about 17-18 h), and then was washed with the PBS solution for three times. After the residual FBS was completely absorbed, 1mL of EGCG diluted by DMEM without serum to corresponding concentration (10 M, 20 M, 50 M, 100 M) was added. After incubation with PK B6 cells at 37 °C for 1 h, it was washed with cold PBS for three times and replaced with cold DMEM without serum, and the cells were infected at 4 °C with porcine pseudorabies virus with the presence of corresponding concentration of EGCG (10 pM, 20 M, 50 M, 100 jM). After incubation for 1 h, it was replaced with 2mL of DMEM nutrient solution with 2% of FBS for maintaining. Then it was incubated in the incubator at 37 °C and 5% CO2 for 24 h. 24 h after the infection, the supernatant cells were collected (1 mL at -70 °C for later TCID50 experiment). After washing the residue with PBS for 3 times and the residual solution was completed absorbed, 2xprotein loading was added to collect the cell sample. It was cooked at 96 °C in the metal bath for 15 min, then the Western blot test was carried out. The results were shown in A in Figure 4. It was found that EGCG decreased the expression of gE and UL42 protein of porcine pseudorabies virus, which confirmed that EGCG decreased the adsorption of porcine pseudorabies virus.

(2) Effect of epigallocatechin gallate measured by TCID50 on the adsorption of porcine pseudorabies virus in PK-15B6 cells

Vero cell digestion was diluted with DMEM nutrient solution with 8% of FBS, and was dropwise added to 96-hole plate at the concentration of 2x103 /hole, placed in the incubator at 37 °C, 5% C02 incubator until the cells attached the wall to form the single layer, then washed with PBS for three times. After the residual solution was completely absorbed, the virus solution of different concentration diluted by DMEM without serum was added, each concentration repeated for 8 times. 1.5 h after the infection, it was replaced with the DMEM with 2% of FBS for maintaining, and the cytopathy situation was observed 72 h after the infection. It was found in B in Figure 4 that EGCG decreased the viral titer of porcine pseudorabies virus infection, which confirmed that EGCG decreased the adsorption of porcine pseudorabies virus.

(3) Effect of epigallocatechin gallate measured by fluorescence quantitative PCR on the adsorption of porcine pseudorabies virus in PK-15B6 cells

PK-15B6 cell digestion was diluted with DMEM nutrient solution containing FBS with concentration of 4% to proper concentration, and was dropwise added to the 6-hole plate at the concentration of 5x10 5/pore, placed in the incubator at 37 °C and 5% of C02 until the cells attached the wall to form the single layer (about 17-18 h), and then was washed with the PBS solution for three times. After the residual FBS was completely absorbed, 1mL of EGCG diluted by DMEM without serum to corresponding concentration (10 M, 20 M, 50 M, 100 M) was added. After incubation with PK-15B6 cells at 37 °C for 1 h, it was washed with cold PBS and replaced with cold DMEM without serum, and the cells were infected at 4 °C with porcine pseudorabies virus with the presence of corresponding concentration of EGCG (10 M, 20 M, 50 M, 100 [M). After incubation for 1 h, it was washed with washed with PBS and absorbed completely. 800OL of PBS was added to scrap the cells with the cell scraper. The cell sample was collected (without repeated freeze thawing) and DNA was extracted for measurement of fluorescence quantitative PCR. The results were shown in C in Figure 4. It was found that EGCG reduced the nucleic acid copy number of porcine pseudorabies virus, which confirmed that EGCG reduced the adsorption of porcine pseudorabies virus.

EMBODIMENT 9

Effect of epigallocatechin gallate on endocytosis of porcine pseudorabies virus (Western blot, fluorescent quantitative PCR and TCID50 test)

(1) Effect of epigallocatechin gallate measured by Western blot on the endocytosis of porcine pseudorabies virus in PK-15B6 cells

PK-15B6 cell digestion was diluted with DMEM nutrient solution containing FBS with concentration of 4% to proper concentration, and was dropwise added to the 6-hole plate at the concentration of 5x10 5/pore, placed in the incubator at 37 °C and 5% of CO2 until the cells attached the wall to form the single layer (about 17-18 h), and then was washed with the PBS solution for three times. After the residual FBS was completely absorbed, 1mL of cold DMEM without serum was added and the cells were infected with porcine pseudorabies virus. After incubation at 4 °C for 1 h, it was washed with cold PBS for three times and the residual solution was completely absorbed. 1mL of cold DMEM with 2% of FBS with the presence of corresponding concentration of EGCG (10 M, 20 M, 50 M, 100 M) was added. It was washed with citric acid for three times and then washed with PBS solution for three times. Then it was replaced with 2mL of DMEM nutrient solution with 2% of FBS for maintaining, then incubated in the incubator at 37 °C and 5% CO2 for 24 h. 24 h after the infection, the supernatant cells were collected (1 mL at -70 °C for later TCID50 experiment). 2xprotein loading was added to collect the cell sample. It was cooked at 96 °C in the metal bath for 15 min, then the Western blot test was carried out. The results were shown in A in Figure 5. It was found that EGCG decreased the expression of gE and UL42 protein of porcine pseudorabies virus, which confirmed that EGCG decreased the endocytosis of porcine pseudorabies virus.

(2) Effect of epigallocatechin gallate measured by TCID50 on the endocytosis of porcine pseudorabies virus in PK-15B6 cells

Vero cell digestion was diluted with DMEM nutrient solution with 8% of FBS, and was dropwise added to 96-hole plate at the concentration of 2x103 /hole, placed in the incubator at 37 °C, 5% C02 incubator until the cells attached the wall to form the single layer, then washed with PBS for three times. After the residual solution was completely absorbed, the virus solution of different concentration diluted by DMEM without serum was added, each concentration repeated for 8 times. 1.5 h after the infection, it was replaced with the DMEM with 2% of FBS for maintaining, and the cytopathy situation was observed 72 h after the infection. It was found in B in Figure that EGCG decreased the viral titer of porcine pseudorabies virus infection, which confirmed that EGCG decreased the endocytosis of porcine pseudorabies virus.

(3) Effect of epigallocatechin gallate measured by fluorescence quantitative PCR on the endocytosis of porcine pseudorabies virus in PK-15B6 cells

PK-15B6 cell digestion was diluted with DMEM nutrient solution containing FBS with concentration of 4%, and was dropwise added to the 6-hole plate at the concentration of 5x10 5/pore, placed in the incubator at 37 °C and 5% of C02 until the cells attached the wall to form the single layer (about 17-18 h), and then was washed with the cold PBS solution for three times. After the residual FBS was completely absorbed, 1mL of cold DMEM without serum was added and the cells were infected with porcine pseudorabies virus. After incubation at 4 °C for 1 h, it was washed with cold PBS for three times and the residual solution was completely absorbed. 1mL of cold DMEM with 2% of FBS with the presence of corresponding concentration of EGCG (10 [M, 20 M, 50 M, 100 [M) was added. After incubation at 37°C for 1 h, it was washed with citric acid for three times and then washed with PBS solution for three times and the residual solution was completely absorbed. 800tL of PBS was added to scrap the cells with the cell scraper. The cell sample was collected (without repeated freeze thawing) and DNA was extracted for measurement of fluorescence quantitative PCR. The results were shown in C in Figure 5. It was found that EGCG reduced the nucleic acid copy number of porcine pseudorabies virus, which confirmed that EGCG reduced the endocytosis of porcine pseudorabies virus.

EMBODIMENT 10

Effects of epigallocatechin gallate on replication of porcine pseudorabies virus (Western blot)

PK-15B6 cell digestion was diluted with DMEM nutrient solution containing FBS with concentration of 4%, and was dropwise added to the 6-hole plate at the concentration of 5x10 5/pore, placed in the incubator at 37 °C and 5% of CO2 until the cells attached the wall to form the single layer (about 17-18 h), and then was washed with the PBS solution for three times. After the residual FBS was completely absorbed, 1mL of cold DMEM without serum was added and the cells were infected with porcine pseudorabies virus. It was incubated in the incubator at 37 °C and 5% CO2 for 1 h, then washed with PBS for three times and the residual solution was completely absorbed. 2mL of DMEM with 2% of FBS with the presence of corresponding concentration of EGCG (10 M, 20 M, 50 M, 100 M) was added. The cell sample was collected 4h and 6h after the infection, and then the Western blot test was carried out. The results were shown in Figure 6. It was found that EGCG decreased the expression of gE and UL42 protein of porcine pseudorabies virus, which confirmed that EGCG decreased the replication of porcine pseudorabies virus.

EMBODIMENT 11

The direct killing effect of epigallocatechin-3-gallate on porcine pseudorabies virus

The PBS or EGCG (10tM, 20[M, 50[M, 100 M) solution was incubated with PRV at 37 °C for 1 h, then inoculated with the PK-15B6 cells for 1 h, and then washed with PBS for 3 times. It was replaced with 2% DMEM, then the protein samples were collected 24 hours later, and the changes of porcine pseudorabies virus protein were detected with Western blot. The above incubated virus was used as TCID50 to detect the variation of porcine pseudorabies virus titer. Results were shown in Figure 7. It was found that EGCG reduced the virus titer of porcine pseudorabies virus infection, which confirmed that EGCG could kill porcine pseudorabies virus directly.

EMBODIMENT 12

Effects of epigallocatechin gallate on swine pseudorabies virus infection in mice

The 6-week-old female mice were divided into 7 groups: healthy control group of 6 mice (intraperitoneal injection of corresponding amount of PBS), dosing control group of 6 mice (intraperitoneal injection of high-dose medicines), infection control group of 6 mice (intraperitoneal injection of corresponding amount of PBS), 4 days in advance of dosing group (low-dose 6, high-dose 6), 2 days in advance of dosing group (low-dose 6, high-dose 6), and 1 hour after infection of dosing group (low-dose 6, high-dose 6). EGCG concentration was 20 mg/ml, low dose of 20 mg/kg per mouse, high dose of 40mg/kg per mouse. The infection dosage of PRV was X10 4 TCID50. The mice of the infection group and 4 days in advance of dosing group were subject to intraperitoneal injection with EGCG for 4 days before the infection, while the mice in other groups were given the corresponding amount of PBS for 4 days and infected 4 days later. The mice of 2 days in advance of dosing group was subject to intraperitoneal injection for 2 days before infection, and infected after 2 days of dosing, and continued dosing for 2 days after infection; The mice in 1 hour after infection of dosing group were dosed 1 h after infection, and the dosing continued for 4 days. After infection, the death of each group of mice per day was counted. The results showed that all the mice in the infection group died on the 7h day, while mice in 4 days in advance of dosing group (low dosing), and 2 days in advance of dosing group (low dosing) had 4 survival and 2 death, 1 hour after infection of dosing group had 3 survival and 3 death, and healthy control group, dosing control group, 4 days in advance of dosing group (high dosing), and 2 days in advance of dosing group (high dosing) had no death (Figure 8). The results showed that EGCG (40 mg/kg) could provide complete protection against porcine pseudorabies virus infection, and it was found that EGCG (40 mg/kg) had effect on the prevention and treatment of porcine pseudorabies virus infection.

As can be seen from the above embodiments, the effect of the epigallocatechin gallate on the infection of porcine pseudorabies virus is very significant, which can reduce the cytopathy caused by porcine pseudorabies virus, inhibit the adsorption, endocytosis and replication of porcine pseudorabies virus, and kill the porcine pseudorabies virus directly. More importantly, epigallocatechin gallate can prevent and/or treat mice infected with porcine pseudorabies virus.

While the foregoing are merely preferred embodiments of the present invention, it should be noted that numerous modifications and adaptations may be made by those of ordinary skill in the art without departing from the principles of the present invention, which are also to be considered as being within the scope of the present invention.

Sequence Listing

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<120> Application of EGCG in the preparation for the prevention and/or treatment of the PRV infection, preparation for the prevention and/or treatment of PRV infection.

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Claims

1. Application of the epigallocatechin gallate in the preparation of the prevention and/or treatment for the infection of porcine pseudorabies virus.

2. The application as described in Claim 1 is characterized in that the epigallocatechin gallate prevents and/or treats the porcine pseudorabies by inhibiting the adsorption, endocytosis and replication of porcine pseudorabies virus.

3. The application as described in Claim 1 is characterized in that the epigallocatechin gallate prevents and/or treats porcine pseudorabies by directly killing the porcine pseudorabies virus.

4. A preparation for the prevention and/or treatment of porcine pseudorabies virus infection is characterized in that the concentration of epigallocatechin gallate in the preparation is 10~100 p mol/L.

5. The preparation as described in Claim 5 is characterized in that the concentration of epigallocatechin gallate in the medicine is 20~80 P mol/L.

6. The preparation as described in Claim 5 is characterized in that the concentration of epigallocatechin gallate in the medicine is 40~60 P mol/L.

7. The preparation as described in Claim 5 is characterized in that the solvent for the medicine is the PBS buffer solution or DMSO.

8. The preparation as described in Claim 8 is characterized in that the concentration of the PBS buffer solution is 0.05~0.15 mol/L.

9. The preparation as described in Claim 9 is characterized in that the concentration of the PBS buffer solution is 0.08~0.12 mol/L.

10. The preparation as described in any of Claims 5~10 is characterized in that the preparation is obtained by dissolving the epigallocatechin gallate in the solvent.

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