JPH01165528A - Attenuated virus of transmissible gastroenteritis of swine, its preparation and use thereof - Google Patents

Abstract

PURPOSE:To obtain an attenuated virus of TGE, resistant to proteinase and/or acid and proliferable in intestines after nasal or oral administration, by the mutagenic treatment of attenuated virus of TGE. CONSTITUTION:An attenuated virus of transmissible gastroenteritis of swine (TGE) is produced by the serial subculture (about 100-180 generations) of a pathogenic TGE virus on a cultured cell of swine. The attenuated virus is subjected to mutagenic treatment such as ultraviolet ray treatment, X-ray treatment or nitrosoguanidine treatment and a virus clone is separated by immediate plaque cloning. Each of the clone is proliferated on a cultured cell of swine, the obtained virus liquid is treated with a proteinase and/or acid and inoculated to a monolayer culture medium for cultured swine cell to effect recovery and proliferation of survived virus. The proteinase or acid treatment and the recovery and proliferation operation of the survived virus are repeated for at least 12 generations to obtain an attenuated TGE virus which can be proliferated on a mucous membrane of intestine.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an attenuated virus that effectively protects newborn piglets from transmissible porcine gastroenteritis (TGE), a method for producing the same, and uses of this virus, ie, a vaccine.

[Background of the invention]

TGE infects pigs of all ages, from suckling pigs to adults, and causes gastroenteritis characterized by watery diarrhea, dehydration, and vomiting, as well as rapid weight loss, growth arrest, and cessation of lactation in sows. In suckling pigs, the younger the age, the higher the mortality rate.
Most of the pigs die within the day, causing severe damage to the pig farming industry.

However, attempts to develop a prophylactic vaccine against TGE
This is not an easy task because E. is a disease caused by a special mode of infection, namely, the proliferation of a pathogenic virus locally in the intestinal tract. In other words, it has been suggested that both neutralizing antibodies in the blood and secreted immunoglobulin A (IgA) are involved in preventing TGE. One of the reasons for this is thought to be that the inoculated attenuated, habituated virus cannot reach the intestinal tract of the sow (Annales de Recherches Ves.
terinaires15, 359-364.1984
). The biggest possibility that adapted attenuated viruses cannot reach the intestinal tract is that these attenuated viruses have lost their resistance to digestive enzymes such as trypsin and acid resistance, which are possessed by the originally pathogenic viruses, due to repeated passages for attenuation. It's something you lose during your teenage years.

[Summary of the invention]

The present invention provides a TGE attenuated virus that has been carefully devised so that a safe TGE attenuated virus from which pathogenicity has been removed does not lack the ability to proliferate locally in the intestinal tract, which is considered necessary for it to be an effective TGE vaccine virus, and a method for producing the same. It is related to.

In summary, the TGE-attenuated virus according to the present invention is resistant to proteolytic enzymes and/or acids, and can proliferate in the intestinal tract after nasal and oral administration, or intranasal or oral administration.

Furthermore, the method for producing an attenuated TGE virus according to the present invention consists of attenuating a pathogenic TGE virus, treating it with a mutagen, and then selecting a protease- and/or acid-resistant strain. .

Furthermore, the TGE prophylactic vaccine according to the present invention comprises an attenuated TGE virus that is resistant to proteolytic enzymes and/or acids that can proliferate in the intestinal tract after nasal and oral administration, or intranasal or oral administration. It is a thing.

Effect The TGE-attenuated virus according to the present invention is usually obtained by attenuating a pathogenic virus by successive passages; Considering that the TGE-attenuated virus according to the present invention was not effective in preventing TGE (detection of neutralizing antibodies in the blood and acquisition of intestinal proliferative ability) It can be said that this was unexpected.

The attenuating process of the TGE attenuated virus according to the present invention is usually carried out through repeated passages. However, in this case, when screening for proteolytic enzyme and/or acid-resistant strains, mutagenic treatment (for example, UV irradiation) improves the efficiency of selecting resistant strains. This effect of mutagen treatment can also be said to be unexpected.

[Detailed description of the invention]

1. Enteropropagating TGE Attenuated Virus The TGE attenuated virus according to the present invention contains at least one of a proteolytic enzyme and/or an acid that can proliferate in the intestinal tract after nasal and oral administration, or nasal or oral administration. It is resistant to

Examples of the protease in this case include trypsin, α-chymotrypsin, pepsin, and others.

These may be a mixture. In addition, each of these proteases, whether used alone or in a mixture, is preferably substantially pure; Good too.

Preferably, the TC,E attenuated virus according to the invention is also acid resistant. Various acids may be used in this case, but hydrochloric acid is the most typical.

The fact that the TGE attenuated virus according to the invention is resistant to at least one of proteolytic enzymes and/or acids means that the TGE-attenuated virus according to the invention is resistant to at least one of proteolytic enzymes and/or acids at temperatures encountered in the stomach or intestinal tract of pigs (e.g.
．． 6° C.) and proteolytic enzymes [e.g. up to 0.5% by weight of trypsin (1:250)] and/or are substantially indestructible under conditions of acidity (e.g. pH 3 to pH 7) (e.g. trypsin (1:250)). This means that the number of viruses destroyed by the resistant strain is 102 or more compared to the susceptible strain.

2. Production of enterically proliferating TGE attenuated virus (1) General explanation The method for producing attenuated TGE virus according to the present invention basically involves attenuation of pathogenic TGE virus by repeated passage on cultured pig cells; It consists of mutagen treatment of the obtained attenuated strain, and selection of proteolytic enzyme and/or acid-resistant strains of the mutagen-treated attenuated strain.

That is, more specifically, pathogenic or highly toxic T.
GE viruses, such as TGE virus KA strain, To
strains, etc., on cultured pig cells, such as cells of kidneys, Suimaru, and other organs, for example, 100 to 18
It becomes attenuated when it is passaged for about 0 generations. At this point, the overwhelming majority of the attenuated strains produced are sensitive to various digestive enzymes and are also acid sensitive. This is subjected to a mutagenic treatment, such as an ultraviolet (UV) treatment, an X-ray treatment, a nitrosoguanidine treatment, and in particular a UV treatment.

The UV treatment can be carried out using known methods such as Vjrology.
17°511-519.1982 and Virolog
y 52, 57-71.1973. After such mutagen treatment, viral clones are usually isolated immediately by plaque cloning. Each of these clones is propagated on cultured pig cells, such as pig kidney cells (CPK cells), and subjected to the step of selecting digestive enzyme and/or acid resistant strains. This sorting process is
It consists of two operations: treatment of the virus fluid with digestive enzymes and/or acid, and after this treatment, the surviving virus is collected and propagated using pig cultured cells. Digestive enzyme and/or acid treatments include
Mixtures of various proteolytic enzymes can be used, such as trypsin, alpha-chymotrypsin, pepsin or vancreatin. Dilute hydrochloric acid is preferred for acid treatment. These enzyme or acid treatments can be performed, for example, at 25°C to 38°C.
C., preferably 37.degree. C., for 10 to 60 minutes. Immediately after the treatment, the mixture is cooled with water, and more preferably calf serum is added to the virus solution to suppress the reaction. This treated virus solution is immediately inoculated into the monolayer culture of the pig cultured cells described above, and the remaining virus is recovered and propagated. For those in which proliferation of residual virus was confirmed, that is, cytopathic effect (CP)
For those in which E) has appeared, after the CPE has sufficiently spread, the virus fluid is collected, preferably subjected to the above-mentioned protease and/or acid treatment, and recovery and propagation of the remaining virus using pig cultured cells. Repeat. This selection procedure is repeated for at least 12 generations to obtain attenuated TGE viruses that are resistant to proteolytic enzymes and/or acids, and therefore are thought to be able to grow on the intestinal mucosa.

(2) Description of preferred specific examples Parent strains include, for example, ferroelectrics isolated from the field, TG
E virus KA strain (llarada et al.

NaLl, 1nsL, Anim, +11th,
QuarL, 7, 127-H7゜1967) can be used. This strain can be obtained from the Ministry of Agriculture, Forestry and Fisheries Biological Resources Research Institute.

Porcine kidney culture (SK) is preferably used for passaging the virus.
The cells were created by digesting healthy pig kidneys with trypsin.
It can be prepared by culturing in Eagle's MEM medium supplemented with 10% calf serum.

The SK cells are inoculated with the diluted virus solution using N and subjected to repeated passage at 37°C. SK until 172s
Virus obtained by passage in cells, namely KA172
As a result of nasal and oral inoculation of the strain virus to four susceptible pigs, as shown in Table 1, the inoculated pigs showed no clinical symptoms such as diarrhea and were normal, and the KA172 strain did not tolerate supplemental milk. It has been proven that the virus has been weakened to the point that it is not pathogenic to pigs. After the attenuated KA172 strain is treated with ultraviolet light (UV), the virus is treated with trypsin at each passage and inoculated into CPK culture cells to carry out serial passage. Passage 12
Examining the trypsin sensitivity of the subsequent viruses reveals that the target trypsin-resistant attenuated TG and E viruses have been created.

Example 1 - After UV treatment of the attenuated KA172 virus (parent strain attenuated KA172 strain) obtained by passage in SK cells,
The cells were trypsinized and subcultured into CPK culture cells.

For UV treatment, put a thin layer of virus solution in a petri dish and apply approximately 400
This is performed by irradiating for 30 seconds at an ultraviolet radiation intensity of μw/ci, and immediately black cloning is performed using CPK cultured cells (pig kidney cell line). C
PK culture cells were treated with 0.25% trypsin solution and 0,2
96 EDTA (ethylenediaminetetraacetic acid disodium trihydrate) solution is added to disperse the cells to form a cell suspension. Next, the cells in this cell suspension are precipitated by centrifugation, and the precipitated cells are taken out to prepare a CPK cell suspension in a cell growth culture medium. The cell growth culture medium used here was Eagle ME
Add 10% calf serum and 10% tryptose phosphate broth by volume to M medium, plus kanamycin 100 μz/ml and fungison 1 μg/ml.
ml (anti-fungal agent). The CPK cell suspension is dispensed into petri dishes and cultured at 37°C for 2 to 3 days. The culture solution is removed from the Petri dish in which the tIt layer of cultured cells has been completely formed, and the virus solution irradiated with UV is inoculated. After adsorbing the virus in a 002 incubator at 37°C for 60 minutes, the virus solution was removed by suction, and the cell growth solution (Tazoshi bovine serum reduced to 5%) was used.
layered with agar solution containing 1% bacto agar,
Further, the sample was incubated at 37° C. for 2 to 3 days, and the sample on which a clear black color was formed was collected and subjected to black cloning.

5 to the cloning virus solution grown in CPK cultured cells.
Trypsin (1:25
0) was added, treated at 37°C for 60 minutes, immediately cooled on ice, and the calf serum was added in equal amounts to the virus solution to suppress the action of trypsin, and then inoculated into CPK cultured cells. These CPK cultured cells are obtained by dispensing the above CPK cell suspension into cell culture bottles and culturing them at 37°C for 2 to 3 days until monolayer cells are completely formed.The culture solution is removed and the virus solution is inoculated. do. After adsorbing the virus at 37°C for 60 minutes, the virus solution is removed by suction, Eagle's MEM medium is added, and culture is carried out at 37°C. After CPE is confirmed (
Usually 2-3 LI after inoculation, the virus is collected. For repeated passage of the virus, add trypsin to the virus solution at 500 to 50%.
000 μg/ml and inoculated into CPK cultured cells in the same manner as the trypsin treatment described above, and the subculture is repeated. When the virus that had been passaged for 12 generations was examined, a TGE virus strain that was trypsin resistant and attenuated was created.

Comparative test 1 Trypsin-resistant attenuated strain obtained above and parent strain attenuated KA1
The trypsin sensitivity of 72 strains was compared. The results are shown in Figure 1 below. Trypsin (1:250) 50
00 μg/ml (37°C.

60 minutes), strain KA172 had a decrease in infectivity of 103 and was susceptible to trypsin, while the attenuated strain resistant to trypsin had a decrease of about 10°''5 and was resistant to trypsin. , the difference in survival rate between the two is 102"7
It was 5.

Comparative Test 2 Regarding the above trypsin-resistant strain and the attenuated parent strain KA172, α-chymotrypsin, pepsin (1:10000
) and vancreatin. The results are as shown in Figure 2.3.4 below.
The attenuated strain is more resistant to trypsin than the other two strains, for example, at 11,000 cz/ml of α-chymotrypsin (37°C, 60°C).
) at pH 4.2, and pepsin at pH 4.2 at pH 4.2.
5, 10 at 500 μg/ml of pushin, 10 at 5000 μg/ml of Hanocreatine (1:10000)
1.0, showing a high survival rate in all cases, and the trypsin-resistant attenuated strain was more resistant than the KA172 strain.

Comparative Test 3 The above trypsin-resistant attenuated strain and the parent attenuated strain KA172 were compared in acid sensitivity when treated at 37° C. for 40 minutes. As shown in Figure 5, the results showed that the KA172 strain had p
There was a decrease in infectivity of 1o3.5 at pH 3.0 and pH 2.0, but for the trypsin 0.5 resistant attenuated strain, the infectivity decreased by about 10 to 10°75 at pH 3.0, and at pH 2. , 0, a decrease in the infectious titer of 103°5 was observed. As a result, the KA172 strain had a pH of 3,
Compared to this, the trypsin-resistant attenuated strain is resistant to pH 3.0 and sensitive to pH 2.0, and the trypsin-resistant attenuated strain has a KA172
It was more resistant than the strain.

Comparative Test 4 The above trypsin-resistant attenuated strain and the parent attenuated strain KA172 were compared in their sensitivity to pig gastrointestinal fluid. The gastrointestinal fluid used was centrifuged at 8000 rpm for 20 minutes. The results are shown in Table 3. Compared to the KA172 strain, the trypsin-resistant attenuated strain was treated with the six contents of the stomach, duodenum, upper, middle, and lower part of the jejunum, and ileum at 37°C for 60 minutes. It was more resistant to these contents.

From the results of Comparative Test Example 1.2.3.4 above, the attenuated strain resistant to I.lipsin is clearly more resistant to trypsin than the attenuated parent strain KA172, and it also contains digestive enzymes such as α-chymotrypsin, pepsin, and pancreatin. It was also demonstrated that it has properties that are more resistant to gastrointestinal fluids, and that it has properties that can be distinguished from the KA172 strain of the parent attenuated TGE virus.

Example 2 Next, in order to examine the safety and effectiveness of the trypsin-resistant attenuated strain, seven susceptible pigs were inoculated nasally and orally with a vaccine made of the trypsin-resistant attenuated strain and observed. 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 anorexia. The strain 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, the trypsin-resistant attenuated strain was inoculated into susceptible pigs, and the distribution of the virus in the body was investigated. The results are shown in Table 2 below. Viruses were recovered from the nasal mucosa, trachea, tonsils, and upper jejunum of the 10th slaughtered pig, and proliferation in the gastrointestinal tract was confirmed, although only in the upper jejunum. In eyes 311 and 51], virus was isolated only from the lungs. No virus was recovered from any other organs tested, gastrointestinal tract, or lymph nodes. These test results demonstrate that the trypsin-resistant attenuated strain has a narrow distribution range in the body, supporting its attenuated virulence and confirming its proliferation in the intestinal tract, which is important as a vaccine virus.

In addition, for pigs inoculated with the above trypsin-resistant attenuated strain, TGE virus To-163 strain (T.

=163 stocks were distributed by the Ministry of Agriculture, Forestry and Fisheries Livestock Hygiene Laboratory. This is an attenuated strain of TGE virus that is widely used as a standard virus for TGE virus neutralization tests and other tests. ), and the production of neutralizing antibodies was confirmed. As shown in Table 1, the neutralizing antibody titer was 128 times to 256 times for pigs inoculated with KA172, and 256 times to 1024 times for pigs inoculated with the trypsin-resistant attenuated strain. It was proven that the antibody response of the vaccinated pigs was as good as or better than that of the immunized pigs.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing trypsin resistance of attenuated strains. Figure 2 is an explanatory diagram showing the α-chymotrypsin resistance of the attenuated strain. Figure 3 is an explanatory diagram showing the pepsin resistance of the attenuated strain. Figure 4 is an explanatory diagram showing the pancreatin resistance of the attenuated strain. This is an explanatory diagram. Figures 5 (a, b) are explanatory diagrams showing the acid resistance of the attenuated strain. Applicant's representative Mr. Sato Figure 1 Figure 2 Figure 4 pH (G) OS< jrensen buffer・Michael
lis buffer ΔMcIIvaine buffer % pH Figure 5

Claims (1)

[Claims] 1. Nasally and orally, or after nasal or oral administration,
Proteolytic enzymes and/or enzymes capable of proliferating in the intestinal tract
An attenuated porcine transmissible gastroenteritis (TGE) virus that is resistant to at least one acid. 2. The TGE attenuated virus according to claim 1, wherein the protease is trypsin. 3. The TGE attenuated virus according to claim 1, wherein the protease is α-chymotrypsin. 4. The TGE attenuated virus according to claim 1, wherein the protease is pepsin. 5. After attenuating the pathogenic transmissible porcine gastroenteritis (TGE) virus, it is treated with a mutagen, and then strains resistant to at least one of proteolytic enzymes and/or acids are selected. A method for producing an attenuated porcine transmissible gastroenteritis (TGE) virus that is resistant to at least one of proteolytic enzymes and/or acids and that can proliferate in the intestinal tract after nasal and oral administration, or intranasal or oral administration. 6. The pathogenic TGE virus is the TGE/KA strain;
A production method according to claim 5. 7. The production method according to claim 5, wherein the attenuation is carried out by repeated passage in cultured pig cells. 8. The production method according to claim 5, wherein the mutagen treatment is ultraviolet irradiation. 9. Claim 5, in which the selection of protease-resistant strains is carried out by trypsin treatment at each successive passage of the virus.
Production method described in section. 10. Attenuated transmissible porcine gastroenteritis (TGE) virus that is resistant to at least one of proteolytic enzymes and/or acids that can proliferate in the intestinal tract after nasal and oral administration, or nasal or oral administration. A vaccine for preventing porcine transmissible gastroenteritis (TGE) comprising: