JPH07110228B2 - Porcine infectious gastroenteritis attenuated virus, its production method and its use - Google Patents

Description

The present invention relates to an attenuated virus that effectively protects against porcine infectious gastroenteritis (TGE) in newborn piglets, a method for producing the same, and use of the virus, that is, a vaccine.

[Background of the Invention]

TGE infects pigs of all ages, from suckling to adult pigs, causing gastroenteritis characterized by watery diarrhea, dehydration, and vomiting, leading to rapid weight loss, stunting, and lactation in sows. In addition, the younger the age of the pigs, the higher the mortality rate.
Most of the pigs died within the age of the day, which caused great damage to the pig farming industry. However, attempts to develop a preventive vaccine for TGE are not easy because TGE is a disease caused by its special mode of infection, that is, the growth of pathogenic virus in the intestinal tract. In other words, blood neutralizing antibodies and secretory immunoglobulin A (IgA) for TGE prophylaxis
It is suggested that both of the two are involved, and as one of the reasons for the failure to establish the protective defense against suckling immunity by cultured cell-adapted attenuated virus in sows, the inoculated attenuated virus cannot reach the intestinal tract of sows. (Annales de Recherches Veterinaires 15 , 359－36
4, 1984). The greatest possibility that the acclimated attenuated virus cannot reach the local intestinal tract is that these attenuated viruses pass the resistance and acid resistance of the original pathogenic virus against digestive enzymes such as trypsin for successive attenuation. To lose in the middle of your generation.

[Outline of Invention]

The present invention provides a safe TGE attenuated virus from which pathogenicity has been removed, and a TGE attenuated virus devised so as not to lack the growth ability in the intestinal tract that is considered necessary for being an effective TGE vaccine virus, and a method for producing the same. It is about.

In short, the TGE attenuated virus according to the present invention is a protein that can be propagated in the intestinal tract without causing diarrhea in the piglet after administration to the piglet by nasal and oral administration, or nasal administration or oral administration. Those that are resistant to degrading enzymes and / or acids.

In addition, the method for producing an attenuated TGE virus according to the present invention comprises the steps of attenuating a pathogenic TGE virus by multiple passages, mutagen treatment, and subsequent selection of protease and / or acid resistant strains. It will be.

Furthermore, the TGE preventive vaccine according to the present invention is nasally and orally, or nasally, or after oral administration to a piglet, the piglet can grow in the intestinal tract without causing diarrhea. It comprises an attenuated TGE virus resistant to degrading enzymes and / or acids.

EFFECT The TGE attenuated virus according to the present invention is usually obtained by subjecting the attenuated virus to successive passages of a pathogenic virus. According to the present invention, since it was not observed, and therefore effective infection protection was not established.
It can be said that it was unexpected that the attenuated TGE virus exerted a protective effect on TGE (detection of neutralizing antibody in blood and acquisition of intestinal proliferative property).

The TGE attenuated virus according to the present invention usually undergoes its attenuation process by successive passages. However, in that case, mutagen treatment (for example, UV irradiation) is necessary when selecting protease and / or acid resistant strains.
By doing so, the selection efficiency of resistant strains is improved. It can be said that this effect of mutagen treatment was unexpected.

[Specific Description of the Invention]

1. Intestinal Proliferative TGE Attenuated Virus The TGE attenuated virus according to the present invention proliferates in the intestinal tract without causing diarrhea in the intestine after administration to a piglet by nasal and oral administration, or nasal administration or oral administration. It is resistant to at least one of a possible proteolytic enzyme and / or acid.

The proteolytic enzyme in this case includes trypsin, α-chymotrypsin, pepsin, and others. These may be a mixture. Further, these proteolytic enzymes, whether alone or as a mixture, are preferably substantially pure ones, but they are in a state of being mixed with other proteolytic enzymes derived from stomach or intestine. Good.

The TGE attenuated virus according to the present invention is preferably acid resistant. Although various acids can be used as the acid in this case, hydrochloric acid is the most typical.

The resistance of the attenuated TGE virus according to the invention to at least one of proteolytic enzymes and / or acids means that the temperature encountered in the stomach or intestinal tract of pigs (eg 39.6).
℃) and proteolytic enzymes [eg trypsin (1:
250) and / or acidity (eg pH 3 to pH 7) under conditions of substantial resistance to destruction (eg more than 10 ² less virus due to destruction of resistant strains than trypsin sensitive strains). )That's what it means.

2. Production of Attenuated TGE Attenuated Virus (1) General Description The method for producing an attenuated TGE virus according to the present invention is basically based on passage (multipassage) of pathogenic TGE virus on cultured pig cells. A), mutagen treatment of the resulting attenuated strain, and selection of proteolytic enzyme and / or acid resistant strains of the attenuated strain treated with mutagen.

That is, more specifically, a pathogenic or highly virulent TG
When E virus, such as TGE virus KA strain, TO strain, etc., is subjected to successive passages on swine culture cells, such as cells of kidneys, testes, and other organs, for example, about 100 to 180 passages. Is attenuated. At this point, the overwhelming majority of the attenuated strains produced are sensitive to various digestive enzymes and acid. This is subjected to mutagen treatment, such as ultraviolet (UV) treatment, X-ray treatment, nitrosoguanidine treatment, and especially UV treatment. UV treatment is a known method, for example, Virology 17,511-519,1962 and Virology 52,57-7.
It can be performed by the method described in 1,1973. After such mutagen treatment, virus clones are usually isolated immediately by plaque cloning. Each of these clones is once grown on porcine cultured cells, for example, porcine kidney cell lines (CPK cells), and then a digestive enzyme and / or acid-resistant strain selection step is performed. This selection step consists of two operations: digestion enzyme and / or acid treatment of the virus solution and, after this treatment, recovery and propagation of surviving virus by porcine cultured cells. Digestive enzymes and / or acid treatment, various proteolytic enzymes,
For example, a mixture of various enzymes such as trypsin, α-chymotrypsin, pepsin or pancreatin can be used. Dilute hydrochloric acid is preferred for the acid treatment. These enzyme or acid treatments include, for example, 25 ° C. to 38 ° C., preferably 37 ° C.
Do for 60 minutes. Immediately after the treatment, the mixture is ice-cooled, and more preferably calf serum is added in an amount equal to that of the virus solution to suppress the reaction. This treated virus solution is immediately inoculated into the monolayer culture of the above-mentioned swine culture cells, and the residual virus is recovered and propagated. For those in which the growth of residual virus could be confirmed, that is, those in which a cytopathic effect (CPE) had appeared, after the CPE had sufficiently spread, the virus solution was collected, and preferably the above-mentioned proteolytic enzyme and / or The acid treatment and the recovery and multiplication operation of the residual virus using porcine cultured cells are repeated.
This selection procedure is repeated for at least twelve generations to obtain attenuated TGE virus that is resistant to proteolytic enzymes and / or acids and is therefore believed to be able to grow on the intestinal mucosa.

(2) Description of preferable specific examples As a parent strain, for example, a virulent strain isolated from the field, TGE
Virus KA strain (Harada et.al., Natl.Inst.Anim.Hlth.Qu
art., 7 , 127-137, 1967) can be used. This strain can be purchased from the Institute for Biological Resources, Ministry of Agriculture, Forestry and Fisheries.

Pig kidney culture (SK) cells preferably used for virus passage can be prepared by digesting healthy pig kidney with trypsin and then culturing in Eagle MEM medium supplemented with 10% calf serum. . The SK cells are inoculated with the virus solution as it is without dilution, and the cells are passaged at 37 ° C. 17
Virus obtained by successive passages to SK cells up to the second generation, namely K
As a result of nasal and oral inoculation of the A172 strain virus to 4 susceptible pigs, as shown in Table 1, clinical signs such as diarrhea were not observed clinically in the inoculated pigs and the KA172 strain was normal. It was proven to be attenuated to the point that it was not pathogenic to pigs. The attenuated KA172 strain is treated with ultraviolet light (UV), then the virus is trypsinized at each passage and inoculated into CPK cultured cells, and passaged for successive passages. When the virus at passage 12 was examined for trypsin susceptibility, it was revealed that the target trypsin-resistant attenuated TGE virus was produced.

Example 1 Attenuated KA172 virus (parent strain attenuated KA172 strain) obtained by successive passages to SK cells was subjected to UV treatment, then trypsinized and passaged to CPK cultured cells.

For the UV treatment, put the virus solution thinly in a petri dish, about 400 μw / c
Irradiation is carried out for 30 seconds under an ultraviolet radiation intensity of m ² , and immediately, plaque cloning is performed using CPK cultured cells (porcine kidney cell line). 0.25 for CPK cultured cells
% Trypsin solution and 0.2% EDTA (disodium ethylenediaminetetraacetic acid disodium salt) solution are added to disperse the cells to make a cell suspension. Next, the cells are precipitated in the cell suspension by centrifugation, the precipitated cells are taken out, and a CPK cell suspension is prepared with a cell growth medium. The cell culture medium used here is Eagle
The MEM medium was prepared by adding 10% fetal calf serum and 10% tryptose phosphate prose in volume percentage, and further mixing 100 μg / ml kanamycin and 1 μg / ml fungusone (antifungal agent). The CPK cell suspension is dispensed into a petri dish and cultured at 37 ° C for 2 to 3 days. The culture solution is removed from the petri dish in which a monolayer of the cultured cells has been completely formed, and the virus solution irradiated with UV is inoculated. After adsorbing the virus in a CO ₂ incubator at 37 ° C for 60 minutes to remove the virus, the virus solution was removed by suction, and the cell growth solution (only 5% of bovine serum was added).
(Amount reduced to 100%) and an agar solution containing 1% of bactoagar are overlaid, and the mixture is allowed to stand at 37 ° C. for 2 to 3 days to collect those in which a clear black is formed and to perform plaque cloning. Trypsin (1: 250) at a concentration of 5000 μg / ml in the cloning virus solution propagated in CPK cultured cells
, And the mixture is treated at 37 ° C. for 60 minutes and immediately cooled on ice, and calf serum is added in an amount equal to that of the virus solution to suppress the action of trypsin and inoculated into CPK cultured cells. The CPK cultured cells were placed at 37 ° C by dispensing the CPK cell suspension into a cell culture bottle.
After culturing for 2 to 3 days, the monolayer cells are completely formed. The culture solution is removed and the virus solution is inoculated. To 37 ℃
After adsorbing the virus for 60 minutes, the virus solution is removed by suction, Eagle MEM medium is added, and the mixture is incubated at 37 ° C. After CPE is confirmed (usually 2 to 3 days after inoculation), the virus is collected. For successive passages of virus, trypsin is added to the virus solution at a rate of 500 to 5000 μg / ml, CPK cultured cells are inoculated in the same manner as the trypsin treatment, and the passage is repeated. When I examined the virus that was passed for 12 generations,
A TGE virus strain having the property of trypsin-resistant attenuated was created.

Comparative Test 1 Attenuated strain resistant to trypsin and attenuated parent strain KA172 obtained above
The strains were compared for trypsin sensitivity. The results are shown in FIG. 1 below. Trypsin (1: 250) 5000 μg / ml (3
After treatment at 7 ℃ for 60 minutes, the strain KA172 had a decrease in infectious titer of 10 ³ and was sensitive to trypsin, but attenuated trypsin resistance was 10 ^0.25 and was resistant to trypsin. . The difference in the survival rate between the two was ^102.75 .

Comparative test 2 For the above-mentioned trypsin-resistant strain and parent strain attenuated KA172 strain,
The sensitivities to α-chymotrypsin, pepsin (1: 10000) and pancreatin were compared. As a result, as shown in FIG.
10 ^1.25 at 00 μg / ml (37 ℃, 60 minutes), pH 4.2 at pepsin
At 500 .mu.g / ml of pepsin, 10 ^2.25 and pancreatin (1: 10000) at 5000 .mu.g / ml of 10 ^1.0 showed high survival rate in all cases, and the trypsin-resistant attenuated strain was KA17.
It was more resistant than the two strains.

Comparative Test 3 The above-described attenuated trypsin-resistant strain and the attenuated parent strain KA172 strain were compared in acid sensitivity when treated at 37 ° C. for 40 minutes. As a result, as shown in FIG. 5, the KA172 strain had pH 3.0 and pH 2.0.
In, there was a decrease in the infectivity titer of 10 ^3.5, trypsin resistant attenuated strain is reduced in 10 ^{0.5-10 0.75} about infectivity in pH 3.0, observed decrease in 10 ^3.5 infectivity in pH2.0 Was given. As a result, the KA172 strain was sensitive to acids having a pH of 3.0 or less, compared with this, trypsin-resistant attenuated strain was resistant to pH3.0 and was sensitive to pH2.0, and the trypsin-resistant attenuated strain was sensitive to acid. KA17
It was more resistant than the two strains.

Comparative Test 4 The sensitivities of the trypsin-resistant attenuated strain and the parent strain attenuated KA172 strain to the pig stomach preparation solution were compared. The gastrointestinal fluid used was spun down at 8000 rpm for 20 minutes. As a result, as shown in Table 3, compared with the KA172 strain, the trypsin-resistant attenuated strain was treated with 6 contents liquids of stomach, duodenum, upper jejunum, middle and lower jejunum, and ileum at 37 ° C for 60 minutes. It was more resistant to the liquid content.

From the results of Comparative Test Examples 1, 2, 3, and 4, the trypsin-resistant attenuated strain is obviously trypsin-resistant as compared with the parent strain attenuated KA172 strain, and α-chymotrypsin, pepsin,
It is more resistant to digestive enzymes and acids of pancreatin, or gastrointestinal fluid, and is a parent strain of attenuated TGE virus K.
It was proved to have a characteristic distinguishable from the A172 strain.

Example 2 Next, in order to examine the safety and efficacy of the above-mentioned trypsin-resistant attenuated strain, 7 vaccines consisting of the trypsin-resistant attenuated strain were intranasally and orally inoculated into 7 susceptible pigs and observed. As a result, as shown in Table 1 below, all the pigs vaccinated with the trypsin-resistant attenuated strain vaccine were normal with no clinical abnormalities such as diarrhea, vomiting, and loss of appetite. Was attenuated to the extent that it was not pathogenic to suckling pigs, and the safety of the trypsin-resistant attenuated strain was confirmed.

In addition, a trypsin-resistant attenuated strain was inoculated into susceptible pigs to examine the distribution of the virus in the body. The results are shown in Table 2 below. The virus was recovered from the nasal mucosa, trachea, flatulence, and upper jejunum of the pigs killed on the 1st day, and it was confirmed that the growth in the digestive tract was only in the upper jejunum. On days 3 and 5, virus was isolated from the lungs only. Other organs tested,
No virus was recovered from the digestive tract or lymph nodes. From these test results, it was confirmed that the trypsin-resistant attenuated strain has a narrow virus distribution in the body, supports the attenuation, and grows locally in the intestinal tract, which is important as a vaccine virus.

In addition, for pigs inoculated with the above-mentioned attenuated trypsin-resistant strain, TGE virus To-163 strain (To-163 strain was distributed from the Ministry of Agriculture, Forestry and Fisheries Animal Health Research Center. As a standard virus for TGE virus neutralization test and other tests. The neutralizing antibody titer against a commonly used attenuated strain of TGE virus was examined and production of the neutralizing antibody was confirmed. The results, as shown in Table 1, are 128 cultures to 256 cultures for the KA172 inoculated pigs.
It was demonstrated that pigs inoculated with the trypsin-resistant attenuated strain had a neutralizing antibody titer of 256 to 1024 times, and that the trypsin-resistant attenuated strain had a better antibody response than the KA172 strain.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing the trypsin resistance of an attenuated strain. FIG. 2 is an explanatory view showing the resistance of the attenuated strain to α-chymotrypsin. FIG. 3 is an explanatory diagram showing pepsin resistance of an attenuated strain. FIG. 4 is an explanatory diagram showing pancreatin resistance of an attenuated strain. FIG. 5 (a, b) is an explanatory view showing acid resistance of the attenuated strain.

Front Page Continuation (72) Inventor Naomi Fukami 2-11-11 Wakamiya, Ichikawa City, Chiba Kenzo Nakayama Dormitory (72) Inventor Minoru Minoru 454-180 Nunokawa, Tone-cho, Kitaoma-gun, Ibaraki Prefecture

Claims (12)

[Claims] 1. An attenuated virus having as a parent strain a virus derived from a KA strain or a TO strain of porcine infectious gastroenteritis (TGE) virus, which has been attenuated by multiple passages, and which is nasally and orally or nasally. , Or after oral administration to a piglet, can grow in the intestinal tract without causing diarrhea in the piglet,
Attenuated TGE virus, which is selected based on the property of being resistant to at least one of protease and / or acid. 2. The attenuated TGE virus according to claim 1, wherein the proteolytic enzyme is trypsin. 3. The attenuated TGE virus according to claim 1, wherein the proteolytic enzyme is α-chymotrypsin. 4. The attenuated TGE virus according to claim 1, wherein the proteolytic enzyme is pepsin. 5. The attenuated TGE virus according to claim 1, wherein the TGE virus is a virus derived from a TEG / KA strain. 6. A proteolytic enzyme and / or which can grow nasally and orally, or after nasal or oral administration to a piglet without causing diarrhea in the intestine.
A method for producing an attenuated porcine transmissible gastroenteritis (TGE) virus that is resistant to at least one acid,
A strain or a virus derived from the TO strain is attenuated by multiple passages, then subjected to mutagen treatment, and then a strain resistant to at least one of a protease and / or an acid is selected. How to make a TGE attenuated virus. 7. The method according to claim 6, wherein the TGE virus is a virus derived from the TGE / KA strain. 8. The production method according to claim 6, wherein the attenuation is carried out by serial passage of cultured cells of pig. 9. The method according to claim 6, wherein the mutagen treatment is ultraviolet irradiation. 10. The production method according to claim 6, wherein the protease-resistant strain is selected by trypsin treatment for each successive passage of the virus. 11. An attenuated virus having as a parent strain a virus derived from a KA strain or a TO strain of porcine infectious gastroenteritis (TGE) virus, which has been attenuated by multiple passages, and which is nasally and orally or nasally. , Or after oral administration to piglets, it is selected based on the property that it can grow in the intestinal tract without causing diarrhea and is resistant to at least one of protease and / or acid. A vaccine for preventing pig infectious gastroenteritis (TGE) for piglets, which comprises an attenuated TGE virus. 12. The preventive vaccine according to claim 11, wherein the TGE virus is a virus derived from a TGE / KA strain.