AU2020100889A4

AU2020100889A4 - An Application Of EGCG In Porcine Epidemic Diarrhea Virus Preparations

Info

Publication number: AU2020100889A4
Application number: AU2020100889A
Authority: AU
Inventors: Qingqing Gao; Song Gao; Tingting GUO; Changchao Huan; Weiyin XU
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2020-05-29
Filing date: 2020-05-29
Publication date: 2020-07-09
Anticipated expiration: 2028-05-29

Abstract

The present invention provides an application of epigallocatechin gallate in porcine epidemic diarrhea virus preparations and relates to the technical field of antiviral application. The epigallocatechin gallate (EGCG) can effectively inhibit the absorption, viropexis, replication, assembly and release of porcine epidemic diarrhea virus so as to inhibit its infection in this invention. Besides, the EGCG can also directly kill the virus, which can be used for the treatment of porcine epidemic diarrhea disease. Drawings of the Specification Mock PEDV PEDV+EGCG (50pM) bt Fig. 1 A 4 8 12 24 36 h.p.i -- + -- + - - + - - + - - + EGCG -+ + -+ + - + + - + + - + + pedv -000li - PEDV-N .. am am "m- maamwactin 14x10 6 B C - + + + + PEDV I12x0. Mock 0 20 50 100 EGCG(gM) I 6 lm PEDV-N 26x404. actionn x10 4 2x1004 0 20 100 Concentration (VM) Fig. 2 1 /'

Description

Drawings of the Specification

Mock PEDV PEDV+EGCG (50pM)

bt

Fig. 1

A 4 8 12 24 36 h.p.i -- + -- + - - + - - + - - + EGCG -+ + -+ + - + + - + + - + + pedv -000li - PEDV-N .. am am "m- maamwactin

6 14x10

B C - + + + + PEDV I12x0. Mock 0 20 50 100 EGCG(gM) I 6

lm PEDV-N 26x404. 4 action x10

2x1004

0 20 100 Concentration (VM)

Fig. 2

1 /'

Descriptions

An Application of EGCG in Porcine Epidemic Diarrhea Virus Preparations

Technical Field The present invention relates to the technical field of antiviral application and in particular to an application of EGCG in porcine epidemic diarrhea virus preparations.

Background Technology Porcine epidemic diarrhea (PED) is a kind of highly contagious intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV), which is characterized by emesis, diarrhea and loss of appetite. Pigs of all ages are susceptible to the disease. The morbidity of infected piglets, feeder pigs and finishing pigs reaches 100%. The disease causes 100% death of neonatal piglets of 10 days old, and finishing pigs to grow slowly and lose weight. In recent years, it has become an epidemic disease in numerous countries around the world and caused huge economic losses to the pig breeding industry due to its high mortality rate.

Porcine epidemic diarrhea virus is a single-strand positive RNA virus with a membrane and belongs to the Coronavirus, the Coronaviridae, the Nidovirales. Studies have found that porcine epidemic diarrhea virus (PEDV) can infect cells of pigs, humans, monkeys and bats. Although no clinical case of human infection has been found, the coronavirus is still an important pathogen that infects humans and animals and constitutes a huge risk to human and animal health. S gene in porcine epidemic diarrhea virus plays a key role in the process of virus adsorption and fusion with host cells, as well as the host chemotaxis. Furthermore, the S gene is prone to mutation, causing it possibly to change its host and infect new species. The transmission between different species therefore may occur, increasing the risk of cross-species infection, thereby threatening human health.

In order to reduce the economic losses arising therefrom and safeguard the health of humans and animals, porcine epidemic diarrhea is prevented in prior art through the method of vaccine immunization, supplemented by enhanced health management and drug treatment. No effective drugs and treatments can be yet provided for porcine epidemic diarrhea.

Descriptions

Invention Summary In view of this, the object of the present invention is to provide an application of EGCG in porcine epidemic diarrhea virus preparations. The epigallocatechin gallate (EGCG) provided by the present invention can effectively inhibit the absorption, viropexis, replication, assembly and release of porcine epidemic diarrhea virus, thereby inhibiting the infection of the virus. Besides, the EGCG can also directly kill the virus, which can be used for the treatment of porcine epidemic diarrhea disease.

In order to achieve the above object of the invention, the technical solutions are provided as follows:

The present invention provides an application of EGCG in porcine epidemic diarrhea virus preparations. Preferably, the preparations include drugs, feed additives, disinfectants and derivatives prepared using EGCG as the skeleton.

The present invention provides an application of EGCG in porcine epidemic diarrhea virus preparations, compared with the prior art, in which the following advantages become apparent:

The EGCG provided by the present invention has a significant effect against the infection of porcine epidemic diarrhea virus. It can reduce the cytopathic effect caused by the virus, inhibit the absorption, viropexis, replication, assembly and release of the virus and directly kill the virus, which provides a reliable scientific and theoretical basis for the treatment of porcine epidemic diarrhea disease.

Brief Description of the Drawings Fig. 1 is a microscope image of the effect of epigallocatechin-3-gallate on the cytopathic effect caused by porcine epidemic diarrhea virus;

Fig. 2 is a graph of inhibition of PEDV infection by epigallocatechin gallate (EGCG), where A and B are Western blot analysis of the effect of epigallocatechin-3-gallate on the expression level of structural protein N of porcine epidemic diarrhea virus in Vero cells; C is the titer of porcine epidemic diarrhea virus in cell supernatant detected by plaque formation

Descriptions

test;

Fig. 3 is a graph of Western blot analysis of the effect of epigallocatechin-3-gallate on the adsorption of porcine epidemic diarrhea virus on Vero cells;

Fig. 4 is a graph of Western blot analysis of the effect of epigallocatechin-3-gallate on the viropexis of porcine epidemic diarrhea virus on Vero cells;

Fig. 5 is a graph of Western blot analysis of the effect of epigallocatechin-3-gallate on the replication of porcine epidemic diarrhea virus on Vero cells;

Fig. 6 is a graph of qRT-PCR analysis of the effect of epigallocatechin-3-gallate on the assembly of porcine epidemic diarrhea virus on Vero cells;

Fig. 7 is a graph showing the effect of epigallocatechin-3-gallate on the release of porcine epidemic diarrhea virus on Vero cells measured by plaque formation test;

Fig. 8 is a graph showing the results of Western blot analysis of the direct killing effect of epigallocatechin-3-gallate on porcine epidemic diarrhea virus.

Detailed description of the Preferred Embodiments The present invention provides an application of EGCG in porcine epidemic diarrhea virus preparations.

In the present invention, the epigallocatechin gallate is also known as epigallocatechin-3-gallate. The source of the epigallocatechin-3-gallate in the present invention is not particularly limited. A conventional commercially available product can be adopted. It is preferably purchased from Selleck. The porcine epidemic diarrhea virus in a embodiment of the present invention is preferably a HLJBY strain. The strain can be a conventional commercially available product.

The preparations in the present invention preferably include drugs, feed additives, disinfectants and derivatives prepared using the EGCG as the skeleton.

Descriptions

In the case that the preparation in the present invention is preferably a drug, the epigallocatechin gallate preferably accounts for 1% to 99% of the total mass of the drug. The excipients of the drug are not particularly limited in the present invention, and the corresponding excipients are preferably selected through the drug dosage form.

In the case that the preparation in the present invention is preferably a feed addictive, the epigallocatechin gallate preferably accounts for 1% to 99% of the total mass of the feed addictive. The auxiliary materials of the feed addictive are not particularly limited in the present invention, and the auxiliary materials for conventional additives are preferred.

In the case that the preparation in the present invention is preferably a disinfectant, the epigallocatechin gallate preferably accounts for 1% to 99% of the total mass of the disinfectant. The auxiliary materials of the disinfectant are not particularly limited in the present invention, and the auxiliary materials for conventionally prepared disinfectants are preferred.

The technical solutions of the present invention will be now clearly and completely described in combination with the embodiments. It is obvious that the embodiments described herein are merely part of the implementations of the present invention, and should not be considered as all the implementations. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the protection scope of the present invention.

Embodiment 1 Effect of epigallocatechin-3-gallate on cytopathic effect caused by porcine epidemic diarrhea virus

Vero cells were diluted with nutrient solution containing 8% fetal calf serum and counted. Targeted cells with a concentration of 5 X105 cells per well were plated into a six-well plate and then were placed into an incubator containing 5% C02 with a set temperature of 37 °C. After they had grown to a density of 70-80% (about 16 hr), the cells were washed twice with PBS solution, pretreated with PBS or EGCG (20 M, 50 M, 100 M) for 1 hr and then were infected with porcine epidemic diarrhea virus HLJBY strain (0.1 MOI). 1 hr later, the solution was changed. At 24 hr of continuous infection, with the drug consistently presented, the morphological changes and cytopathic effect conditions of the cells were observed through the

Descriptions

microscope, as shown in Fig. 1. It can be seen from Fig. 1 that the EGCG can reduce the cytopathic effect caused by porcine epidemic diarrhea virus.

Embodiment 2 Effect of epigallocatechin-3-gallate on expression level of structural protein N of porcine epidemic diarrhea virus in cells and on virus titer in supernatant

Vero cells were diluted with nutrient solution containing 8% fetal calf serum and counted. Targeted cells with a concentration of 5 X105 cells per well were plated into a six-well plate and then were placed into an incubator containing 5% C02 with a set temperature of 37 °C. After they had grown to a density of 70~80% (about 16 hr), the cells were washed twice with PBS solution, pretreated with PBS or EGCG (50 M) for 1 hr respectively and then were infected with porcine epidemic diarrhea virus HLJBY strain (0.1 MOI). 1 hr later, the solution was changed. At 4 hr, 8hr, 12 hr, 24 hr and 36 hr of continuous infection, with the drug consistently presented, the protein samples of the cells were collected and the expression level of structural protein N of porcine epidemic diarrha virus was analyzed by Western blot. As described above, the cells were pretreated with PBS or EGCG (20 M, 50 M, 100 M) for 1 hr respectively and then were infected with porcine epidemic diarrhea virus HLJBY strain (0.1 MOI). 1 hr later, the solution was changed. At 24hr of continuous infection, with the drug consistently presented, the cell supernatant was collected and then the virus titer of the supernatant was detected by plaque formation test. After three times of wash with PBS solution, the protein samples of the cells were collected, and the expression level of structural protein N of porcine epidemic diarrhea virus was analyzed by Westernblot, as shown in Fig. 2. It can be seen from Fig. 2 that the EGCG can significantly reduce the expression level of structural protein N of porcine epidemic diarrhea virus in Vero cells. At the same time, the virus titer of the supernatant is also reduced.

Embodiment 3 Effect of epigallocatechin-3-gallate on infection process of porcine epidemic diarrhea virus

(1) The effect of epigallocatechin-3-gallate on the adsorption of porcine epidemic diarrhea virus (Western blot)

Descriptions

Vero cells were diluted with nutrient solution containing 8% fetal calf serum and counted. Targeted cells with a concentration of 5 X105 cells per well were plated into a six-well plate and then were placed into an incubator containing 5% C02 with a set temperature of 37 °C. After they had grown to a density of 70~80% (about 16 hr), the cells were washed three times with PBS solution, pretreated with PBS or EGCG (20 M, 50 M, 100 M) for 1 hr respectively and then were infected with porcine epidemic diarrhea virus HLJBY strain (0.1 MOI). The infected cells were incubated at 4 °C in a refrigerator for 1 hr with the corresponding drug concentration, and then were washed three times with cold PBS, with the addition of 2% DMEM, and then were placed in the incubator at 37 °C. At 24 hr, the protein samples of the cells were collected, and the expression level of protein N of the virus was detected, as shown in Fig. 3. It can be seen from Fig. 3 that the EGCG has significantly inhibited the adsorption of porcine epidemic diarrhea virus.

(2) The effect of epigallocatechin-3-gallate on the viropexis of porcine epidemic diarrhea virus (Western blot)

Vero cells were diluted with nutrient solution containing 8% fetal calf serum and counted. Targeted cells with a concentration of 5x105 cells per well were plated into a six-well plate and then were placed into an incubator containing 5% C02 with a set temperature of 37 °C. After they had grown to a density of 70 ~ 80% (about 16 hr), the cells were infected with porcine epidemic diarrhea virus HLJBY strain (0.1 MOI) at 4 °C for 1 hr, washed three times with cold PBS solution, and then were treated with PBS or EGCG (20 M, 50 M, 100 M) respectively. The infected cells were incubated at 37 °C for 1 hr, and then were washed three times with citric acid solution and PBS respectively. At 24 hr of continuous infection, the protein samples of the cells were collected, and the expression level of protein N of the virus was detected, as shown in Fig. 4. It can be seen from Fig. 4 that the EGCG has significantly inhibited the process of viropexis of porcine epidemic diarrhea virus.

(3) The effect of epigallocatechin-3-gallate on the replication of porcine epidemic diarrhea virus (Western blot)

Vero cells were diluted with nutrient solution containing 8% fetal calf serum and counted. Targeted cells with a concentration of 5x105 cells per well were plated into a six-well plate and then were placed into an incubator containing 5% C02 with a set temperature of 37 °C. After

Descriptions

they had grown to a density of 70-80% (about 16 hr), the cells were washed three times with PBS solution, and then were infected with porcine epidemic diarrhea virus HLJBY strain (0.1 MOI). 1 hr later, the solution was changed and PBS or EGCG (50 M) solution was added for further treatment. At 4 hr and 6 hr of continuous infection, the protein samples of the cells were collected, and the effect of EGCG on the expression level of structural protein N of porcine epidemic diarrhea virus was detected by Westernblot, as shown in Fig. 5. It can be seen from Fig. 5 that the EGCG can reduce the expression level of structural protein N of porcine epidemic diarrhea virus and then significantly inhibit the replication of the virus.

(4) The effect of epigallocatechin-3-gallate on the assembly of porcine epidemic diarrhea virus (Western blot)

Vero cells were diluted with nutrient solution containing 8% fetal calf serum and counted. Targeted cells with a concentration of 5x105 cells per well were plated into a six-well plate and then were placed into an incubator containing 5% C02 with a set temperature of 37 °C. After they had grown to a density of 70 ~ 8 0 % (about 16 hr), the cells were washed three times with PBS solution, and then were infected with porcine epidemic diarrhea virus HLJBY strain (0.1 MOI). 1 hr later, the solution was changed and PBS or EGCG (20 M and 50 M) solution was added for further treatment. At 24 hr of continuous infection, the cell supernatant was collected, while Trizol was added for the treatment. The cells were washed three times with PBS and treated with Trizol to extract total RNA from the cell supernatant and the cells. cDNA was synthesized by reverse transcription. The expression level of ORF3 gene in the cell supernatant and cells were quantitatively detected by fluorescence, as shown in Fig. 6. It can be seen from Fig. 6 that the EGCG has significantly inhibited the assembly of porcine epidemic diarrhea virus.

(5) The effect of epigallocatechin-3-gallate on the release of porcine epidemic diarrhea virus (plaque formation test)

Vero cells were diluted with nutrient solution containing 8% fetal calf serum and counted. Targeted cells with a concentration of 5x105 cells per well were plated into a six-well plate and then were placed into an incubator containing 5% C02 with a set temperature of 37 °C. After they had grown to a density of 70~80% (about 16 hr), the cells were washed three times with PBS solution, and then were infected with porcine epidemic diarrhea virus HLJBY strain (0.1

Descriptions

MOI). 1 hr later, the solution was changed and PBS or EGCG (20 M and 50 M) solution was added for further treatment. At 24 hr of continuous infection, the cell supernatant was collected and then was washed three times with PBS solution. With the addition of 1 mL of serum-free nutrient solution to each well, the supernatant was frozen and thawed 2 to 3 times repeatedly before it was collected into the EP tube. The titers of porcine epidemic diarrhea virus in the cell supernatant and in the cells were detected by plaque formation test, as shown in Fig. 7. It can be seen from Fig. 7 that the EGCG has significantly inhibited the release of porcine epidemic diarrhea virus.

(6) The direct killing effect of epigallocatechin-3-gallate on porcine epidemic diarrhea virus (Western blot)

PBS or EGCG (50 M and 100 M) solution was incubated with PEDV at 37 °C for 1 hr, then mixed solution inoculation with Vero for 1 hr, then washed three times with PBS, and then was replaced by 2% DMEM. At 24 hr, the protein samples were collected, and the expression level of structural protein N of porcine epidemic diarrhea virus was detected by Western blot, as shown in Fig. 8. It can be seen form Fig. 8 that the EGCG can directly kill porcine epidemic diarrhea virus.

It is evident from the embodiments 1 to 3 that EGCG can reduce the cytopathic effect caused by porcine epidemic diarrhea virus, inhibit the adsorption, viropexis, assembly and release of porcine epidemic diarrhea virus, and can directly kill porcine epidemic diarrhea virus.

The above mentioned should be understood as various preferred embodiments of the present invention only. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and retouches can be made. These improvements and retouches should be also regarded as the protection scope of the present invention.

Claims

1. It is an application of EGCG in porcine epidemic diarrhea virus preparations.

2. The application defined in claim 1 wherein the preparations include drugs, feed additives,

disinfectants and derivatives prepared using EGCG as the skeleton.