JP2000044473A - Livestock virus infection preventive agent - Google Patents

Abstract

(57) [Problem] To provide a drug which effectively inhibits the infection of herpes virus and coronavirus, and which has no fear of causing harmful side effects to the living body. SOLUTION: The present invention provides an agent for preventing herpesvirus or coronavirus infection of livestock, comprising tea polyphenol as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a preventive agent for livestock virus infection, and more particularly to a preventive agent for livestock herpesvirus or coronavirus infection. Further, the present invention relates to a method for preventing livestock virus infection.

[0002]

2. Description of the Related Art Aujeszky's disease is an infectious disease caused by a kind of herpes virus and infects pigs, cows, sheep, dogs, cats, and the like. Non-pig animals die shortly after infection, but pigs are relatively resistant, especially adult pigs, which are often mild or asymptomatic and well tolerated. Bovine infectious rhinotracheitis is an infectious disease caused by infection with the same herpesviridae virus, and is mainly characterized by respiratory symptoms in cattle.

[0003] Various vaccines have been developed as agents for preventing the transmission of these infectious diseases. However, immunization with vaccine (vaccination)
It has been shown that, if done, the disease can be prevented but not the infection. For any infectious disease,
No drug has been found that completely blocks the infection of the causative herpes virus.

[0004] Although adult pigs are relatively resistant to Aujeszky's disease, as described above, pigs that have become resistant to infection can serve as potential carriers to transfer virus or viral genome for a long period of time. It is considered that latent virus is activated and excreted when it is kept and subjected to stress due to environmental factors and becomes a source of infection. Cows infected with bovine infectious rhinotracheitis are also considered to be potential carriers and become the source of infection in the same course as pigs.

[0005] As described above, since it is considered that infection of both infectious diseases cannot be completely prevented by vaccination, the infection of these viruses can be effectively prevented, and furthermore, harmful to living organisms can be prevented. There is a demand for the development of a drug that can be used safely without side effects.

On the other hand, swine infectious gastroenteritis and swine epidemic diarrhea are infectious intestinal infections of swine caused by different types of coronaviruses. Both are infected regardless of age, but younger ones have more severe symptoms and suckling piglets often show higher mortality.

[0007] Preventive measures should be taken for any infectious disease,
It has been implemented to protect suckling pigs from virulent viruses. Specifically, the milk immunization method, that is, immunizing a sow to produce antibodies, mainly IgA antibodies in milk, and drinking the milk to piglets, thereby protecting the intestinal tract surface of the piglets with the antibodies. The way has been done.

However, vaccination by this method has not always been established as a method for sufficiently raising antibodies. In addition, in order to maintain the best immunity at the time of calving of sows with different calving dates, it is extremely complicated because it is necessary to consider the calving date at the time of vaccination.

As described above, even in infectious diseases caused by the above-mentioned coronavirus, the prevention of infection by the provision of immunity is not yet complete. Therefore, development of a drug that can effectively prevent coronavirus infection, has no harmful side effects to the living body, and can be used with confidence is desired.

[0010]

Means for Solving the Problems The present inventors have determined the above-mentioned problems, that is, herpes viruses such as Aujeszky's disease virus and bovine infectious rhinotracheitis virus, and pig infectious gastroenteritis virus and swine epidemic diarrhea virus. The present inventors have conducted intensive studies to effectively prevent coronavirus infection and to develop a drug that does not cause harmful side effects to the living body.

In the present invention, from the viewpoint of placing emphasis on safety for living organisms, research was conducted to search for a substance having a desired medicinal effect from natural products, not from chemically synthesized products. As a result, they have found that tea polyphenols, which are tea extracts, have an action of inhibiting the above-mentioned virus infection, and have reached the present invention based on such findings.

The present invention according to claim 1 is a preventive agent for herpesvirus or coronavirus infection of livestock, comprising tea polyphenols as an active ingredient. The present invention according to claim 2 is the virus infection preventive agent according to claim 1, wherein the herpes virus is Aujeszky's disease virus or bovine infectious rhinotracheitis virus. According to a third aspect of the present invention, the coronavirus is a swine infectious gastroenteritis virus or a swine epidemic diarrhea virus.
The prophylactic agent for viral infection according to the above. The present invention according to claim 4 is a method for preventing viral infection of livestock, which comprises administering the agent for preventing viral infection according to claim 1 to livestock in addition to feed or drinking water of the livestock. The present invention according to claim 5 is a method for preventing virus infection in livestock, which comprises spraying the agent for preventing virus infection according to claim 1 onto the skin of livestock.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The infection preventive agent of the present invention contains tea polyphenols as an active ingredient. Tea polyphenols are substances contained in tea. Here, tea means tea tree (Came
llia sinensis), leaves, stems, xylem, roots, nuts or mixtures thereof, but drinking tea leaves are common. There are various types of drinking tea leaves depending on the manufacturing method.
For example, fermented teas such as black tea, Pu'er tea, semi-fermented teas such as oolong tea, wrapping tea, green tea, pot-roasted green tea, unfermented teas such as roasted tea, and mixtures thereof. These tea leaves all contain tea polyphenols.

In the present invention, tea itself can be used as the tea polyphenols, but a tea extract obtained by extracting tea with water, hot water, an organic solvent, a water-containing organic solvent or the like is preferably used. Further, a tea polyphenol-rich material obtained by further purifying the tea extract to a desired degree using an organic solvent fractionation or an adsorption resin can also be used. Such extraction and purification methods are described in, for example,
JP-A-44234, JP-A-2-12474, JP-A-2-124
22755, JP-A-4-20589, 5
The methods described in JP-A-260907 and JP-A-8-109178 can be employed.

[0015] Examples of tea polyphenols include catechins and theaflavins. Examples of catechins include (+)-catechin, (−)-catechin,
(+)-Gallocatechin, (+)-epigallocatechin,
(+)-Gallocatechin gallate, (+)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-epicatechin gallate, (-)-catechin gallate, (-)-
Epigallocatechin, (-)-gallocatechin, (-)-epigallocatechin gallate, (-)-gallocatechin gallate and the like. Examples of theaflavins include theaflavin monogallate A, theaflavin monogallate B, theaflavin digallate, and free theaflavin. In the present invention, these tea polyphenols are used alone or in combination of two or more.

Commercially available tea polyphenols can also be used. As a commercially available product containing catechins as a main component, trade name: Polyphenon 60 (manufactured by Mitsui Norin Co., Ltd.
Tea polyphenol content 60% or more), trade name: Polyphenon 30 (Mitsui Norin Co., Ltd., tea polyphenol content 30% or more), trade name: Polyphenon 70S (Mitsui Norin Co., Ltd., tea polyphenol content 70% or more), etc. . Commercial products containing theaflavins as the main component include polyphenone TF (manufactured by Mitsui Norin Co., Ltd.
Composition: theaflavin 16.8%, theaflavin monogallate A 19.5%, theaflavin monogallate B
16.1%, theaflavin digallate 31.4%).

As mentioned above, the viruses to which the agent for preventing viral infection of livestock according to the present invention is applied are herpes virus and coronavirus. Examples of the herpes virus include Aujeszky's disease virus (AD virus) and bovine infectious rhinotracheitis virus (IBR virus) as described in claim 2 of the present invention. Examples of the coronavirus include swine infectious gastroenteritis virus (TGE virus) and swine epidemic diarrhea virus (PED virus) as described in the present invention.

The specific mechanism by which the agent for preventing viral infection of the present invention exerts an effect of preventing infection against these viruses has not yet been elucidated. In general, viruses that infect and multiply animal cells utilize substances (mainly glycoproteins) on cell membranes as receptors at the start of infection. That is, a specific protein (binding) existing on the surface of the virus particle is
protein) specifically binds. The bound virus is taken into the cytoplasm and infection is established. Therefore, it is considered that viral infection can be prevented by suppressing the activity of the binding protein or the activity of the receptor on the cell membrane.

The form of use of the agent for preventing viral infection of the present invention is various and is not limited. However, as described in claims 4 and 5, it can be administered to livestock in addition to livestock feed or drinking water, or can be administered to livestock. The main form is spraying on the skin. As a method of spraying on the skin of livestock, there is a method of mixing a virus infection preventive agent with water (fine fog) sprayed from above a livestock barn or the like. The fine fog is mainly performed for the purpose of lowering the body temperature of livestock such as pigs, which can prevent virus infection and prevent the spread of disease. Therefore,
There is no particular limitation on the dosage form, as long as it contains tea polyphenols as an active ingredient. Generally, it is used in the form of a solution or powder. It should be noted that additives and auxiliary components used for ordinary medicines, for example, diluents, extenders, excipients, and the like can be appropriately used in combination.

In the prophylactic agent for virus infection of the present invention, the amount of the tea polyphenols, which is an active ingredient, depends on the type of virus to be treated, the dosage form of the drug, the age and health of livestock, and the environmental conditions such as livestock barn. It may be appropriately determined in consideration of such factors. For example, when added to livestock feed or drinking water, it is 0.01 to 1.0%, preferably 0.1 to 0.1%.
6% is appropriate. On the other hand, when it is added to water for watering, about 0.01 to 0.6% is appropriate. In addition, since tea polyphenols are components of tea that are consumed on a daily basis, there is no concern about livestock safety. Therefore, it is possible to administer more than the above-mentioned amount and to administer continuously for a long period of time.

[0021]

Next, the present invention will be described with reference to examples. The virus and tea extracts used in the examples are as follows. a) Virus ・ Herpes virus Aujeszky's disease virus (AD virus) Bovine infectious rhinotracheitis virus (IBR virus) ・ Corona virus Swine infectious gastroenteritis virus (TGE virus) Swine epidemic diarrhea virus (PED virus)

B) Tea extract Trade name: Polyphenone 70S (manufactured by Mitsui Norin Co., Ltd.) dissolved in PBS to a concentration of 10 mg / ml.

C) Cells used for culture Different cells were used for each virus. -AD virus, TGE virus: Pig kidney-derived cell line (CPK cell)-IBR virus: Bovine kidney-derived cell line (MDBK cell)-PED virus: Monkey kidney-derived cell line (Vero cell) These cells Used in the test was grown in a cell growth medium (Eagle's medium containing serum and tryptose phosphate broth).

Example 1 Infectivity of Herpesvirus Treated with Tea Extract Whether or not infectivity could be prevented by treating herpesvirus with a tea extract was examined. 0 tea extract
μg / ml, 0.1 μg / ml, 0.3 μg / ml, 1
It was dissolved in PBS to have a concentration of μg / ml, 3 μg / ml, and 10 μg / ml. On the other hand, it was diluted with PBS herpesvirus (AD virus, IBR virus) so as to have a 100 TCID _50. Here, 100 TCID ₅₀ indicates the amount of the virus, and means 100 times the amount of the virus that causes infection in 50% of the cultured cells.

Herpes virus (AD virus, IBR virus) was added to the above-mentioned various concentrations of tea extract and treated for 30 minutes, and the resulting treatment solution of each virus was inoculated into cell culture wells and incubated at 37 ° C. for 5-7 minutes. Cultured for days. Observing cell culture with an optical microscope, CPE as an indicator of viral infection
The presence or absence of (cytopathic effect) was examined, and the infectivity inhibition rate was calculated by the following equation. The CPE is a morphological change of a cultured cell caused by the propagation of a virus, and is the most direct method for determining whether or not the virus has grown in a cell culture. The relationship between the dose of the tea extract and the inhibition rate of virus infectivity is shown in FIGS. Table 1 shows the dose of the tea extract that inhibited the virus infection by 100%.

[0026]

(Equation 1)

[0027]

[Table 1]

1 and 2 that the treatment solution of tea extract of each herpes virus loses infectivity to cells as the dose of tea extract increases, that is, the effect of inhibiting infection is dose-dependent. Is evident. As shown in Table 1, treatment with the tea extract at a concentration of 1 μg / ml reduced the infectivity of the AD virus to 10%.
It turned out that 0% can be prevented. Also, similarly, IBR
Virus infectivity was found to be blocked by as little as 0.1 μg / ml of tea extract. Therefore, it can be seen that direct treatment of the herpes virus with the tea extract can prevent the infection, and particularly when the herpes virus is used at a predetermined dose or more, the infection can be completely inhibited by a short treatment.

Example 2 [Effect of Tea Extract Treatment on Pre-infection Cells] It was examined whether or not pre-treatment of cells before infection with a tea extract could prevent herpes virus infection. 100 μg / ml or 300 μg /
A porcine kidney-derived cell line (CPK cell) was immersed in this for 30 minutes. The treated cells were infected with the AD virus, and the presence or absence of the infection was examined.

Further, the cells were used as cell lines derived from bovine kidney (MDB).
K cells) were tested in the same manner as described above, except that the virus was replaced with the IBR virus. Table 2 shows the virus infection inhibition rates (%) obtained as a result of each test.

[0031]

[Table 2]

As is clear from Table 2, it was confirmed that the pretreatment of the cells before infection with the tea extract can effectively prevent herpes virus infection. From this, it was found that the infection preventive agent of the present invention can prevent normal cell infection beforehand.

Example 3 Infectivity of Coronavirus Treated with Tea Extract In Example 1, AD virus was replaced with TGE virus,
The test was performed in the same manner as in Example 1 except that the IBR virus was replaced with a PED virus and the MDBK cells were replaced with Vero cells. The relationship between the dose of the tea extract and the inhibition rate of each virus infectivity is shown in FIGS. In addition, one virus infection
Table 3 shows the doses of tea extract that were inhibited by 00%.

[0034]

[Table 3]

3 and 4 that treatment of each coronavirus with tea extract results in a loss of infectivity of the virus to the cells as the dose of tea extract increases, ie, inhibition of infectivity is dose-dependent. It is clear that there is. Furthermore, as shown in Table 3, when the virus was treated with 10 μg / ml of the tea extract, the infection of each virus was reduced by 1%.
00%. From this, it was revealed that the infection preventive agent of the present invention exhibited an infection inhibitory effect not only on herpes virus but also on coronavirus.

Example 4 [Effect of tea extract treatment on pre-infection cells] In Example 2, AD virus was replaced with TGE virus,
In addition, the IBR virus was replaced with the PED virus, the MDBK cells were replaced with Vero cells, and the concentration of the tea extract was changed to 3%.
The procedure was performed in the same manner as in Example 2 except that 0 μg / ml or 100 μg / ml was used. Table 4 shows the resulting viral infection inhibition rates.

[0037]

[Table 4]

Table 4 shows the effect of the treatment of the pre-infection cells with the tea extract. That is, a sufficient infection inhibition rate cannot be obtained at a concentration of 30 μg / ml for the TGE virus, but a complete infection inhibitory effect is exhibited at a concentration of 100 μg / ml. For PED virus, the concentration is 30μ.
g / ml shows a sufficient infection inhibition rate, and 100 μg / m
1 shows a complete infection inhibitory effect. This indicates that treatment of normal cells with a tea extract in advance shows an excellent infection-inhibiting effect on coronavirus.

[0039]

EFFECT OF THE INVENTION The viral infection preventive agent of the present invention can effectively prevent herpes virus and coronavirus infections when administered in the form of feed or drinking water of livestock. In addition, it may be used for washing fertilized eggs and semen, and also for disinfecting livestock barns, etc., and does not pollute the environment and can prevent infection of the virus at a low dose. In addition, since the active ingredient of this virus infection preventive agent is derived from a natural product that is consumed by humans in a considerable amount on a daily basis, there is no problem in terms of safety when applied to a living body as a drug. There is no adverse effect due to the use of.

[Brief description of the drawings]

FIG. 1 is a graph showing the inhibitory effect of the infection preventive agent of the present invention on infection with AD virus.

FIG. 2 is a graph showing the inhibitory effect of the infection preventive agent of the present invention on infection with IBR virus.

FIG. 3 is a graph showing the inhibitory effect of the infection preventive agent of the present invention on TGE virus infection.

FIG. 4 is a graph showing the inhibitory effect of the infection preventive agent of the present invention on PED virus.

 ────────────────────────────────────────────────── ─── Continued on the front page F term (reference) 2B150 AA01 AA02 AA03 AB10 DB01 DD31 DD42 4C062 FF44 FF56 4C086 AA01 AA02 BA08 MA08 ZB33 ZC61 4C088 AB45 BA08 BA09 ZB33

Claims (5)

[Claims] 1. An agent for preventing herpesvirus or coronavirus infection of livestock, comprising tea polyphenol as an active ingredient. 2. The herpes virus is Aujeszky's disease virus or bovine infectious rhinotracheitis virus.
The agent for preventing viral infection according to the above. 3. The method according to claim 1, wherein the coronavirus is swine infectious gastroenteritis virus or swine epidemic diarrhea virus. 4. A method for preventing viral infection according to claim 1,
A method for preventing livestock virus infection, which comprises administering to a livestock in addition to the feed or drinking water of the livestock. 5. The agent for preventing viral infection according to claim 1,
A method for preventing livestock virus infection, which is sprayed on the skin of livestock.