JPS58502210A - Bluetongue virus vaccine, method for producing the same, and method for immunizing ruminants with this vaccine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention is directed to the use of blue tongue virus+E. Vaccines for immunizing animals against the international serotypes of The present invention relates to a method for producing a vaccine and a method for immunizing ruminant animals using the vaccine.

Bluetongue, a viral disease transmitted by arthropods, affects sheep, cattle, and sheep. and occurs in wild anti-swimming animals. Bluetongue disorders in infected animals are caused by infectious Bovine viral diarrhea, varicella stomatitis, malignant catarrhal fever, fungal stomatitis, cowhide, photosensitization and similar to foot-and-mouth disease.

Bluetongue virus causes encephalitis in cattle, infertility, abortion and maimed birth in cattle and sheep. It causes As something that causes everything from subclinical infections to acute severe infections. , 20 serotypes have been reported in the literature. Chronic, constantly releasing virus A cow was also found. The virus affects endothelial cells, periendothelial cells and capillaries of the vasculature. infects pericytes, pericillary arteries and venules.

See 5tair et al., 5PathologiaVet 164 (1968).

The virus has an affinity for the reticuloendothelial system and can be transmitted into infected sperm. Lued e etc. Proc 2Q thWor14 Vet Congress 2039 (1976).

Although the direct cost of death due to BTV is sometimes high, the indirect cost is much higher for the economy. has a meaning. The physical condition of BTV deteriorates significantly, and the sale of slaughtered animals has become difficult. 5ru. In sheep infected with BTV, wool growth is impaired by hair shedding, resulting in defects. Gives high or low yield wool mass. Significantly weakened as a result of BTV infection , resistance to secondary bacterial or Chlamydomonas infections and other invasive agents. Decrease. The fertility of infected animals is also reduced. Infected animals may have a miscarriage or give birth to a malformed pup. As a result, the animal becomes infertile during one or more breeding seasons. blue tan The most important damage caused by bluetongue infection is that cattle from areas where bluetongue is endemic. , producers who export cow semen and sheep are subject to export bans or strict testing requirements. This is an economic loss due to the imposition.

Under natural conditions, virus transmission is limited to at least four species, Cu1icoj, d, and es. In other words, it happens when you are bitten by a sand fly or a nuka force. By the same Nukaka Befter Experiments have shown that BTV transmission occurs between cattle and sheep.

Lu, ed. Re et al., 28 AJVP, 457 (1967). cattle, sheep and Many wild animals carry BTV, and the virus survives the winter. at least 6 years continues). Persistent BTV viremia has been confirmed in cattle. Hou rrigan'.

51 AustVet J 170 (1975). Once a country has BTV Once inside, it seems impossible to eradicate the virus. Era-smus ”, 51 Au5t Ve also J209 (1975).

It belongs to the genus Orbivirus. This name comes from the naming of the virus in 1971. ＼＼reo1. ike” virus from 0rbiviru s　I/C was changed. The pathogenicity of the bluetongue virus was first discovered in 1906 by The Established by Iler. Erasmus, 51 Aust Vet, J 165 (1975). Since then, (a) cattle, sheep, sheep and and the isolation of bluetongue virus from some wild animals, b) Description of transmission, livestock epidemics, and clinical and pathogenic characteristics of the bluetongue family and (C) Infections resulting from BTV serotypes of rM are described.

An example is DuToit190nderstepoort J Vet SCI Anhm Indus 7 (1944); Komarov and Golds mit, F3 Refuah vat 96 (1951): Pr1cea ndHardy, 124 J Aml: eaAS3n 255 < 1954 ) ; 5l-tope etc., 111 JExp Med 1 5 5 (196 0) ; Ding L I V i n g S -on and Harav.

25 AJVR1958 (1964); L uedxe etc., 30AJVR 511 (1969) ; ) lourrlgan ahci Kllngspo rr+.

See 51 Au5t Vet J 170 (1975).

The immune defenses of bluetongues in the United States are Using the BTV international serotype 10 vaccine, McKerchsr et al. 18 AJVRro 10 (1975), which was attempted with great success. This generation 4 The model of the object is Ale Xander's method (210nderstepoort JVe tSciAnimIndus 231 (1947)). Na Oh, this person was the first to successfully propagate bluetongue virus in chicken embryos. He is a person who

Today, we have an egg-attenuated multivalent live virus vaccine containing several bluetongue strains. use Vaccine processing is routinely carried out in South Africa and Israel .

Produced by Cutter Laboratories and used in the United States. Egg-adapted vaccines have been discontinued from marketing because they cause severe reactions in vaccinated sheep. Ta. Next, Kemeny and Drehle used the 2’2AJVR921 (19 BTV Kokusai Life 010 kidney cell culture from eggs as described in 61). adapted. Produced by Col1orado Serum Company This modified live virus vaccine has been used in sheep.

To date, there have been few reports on vaccination against BTV in cattle. . BTV is an effective vaccine for cattle, as it plays an important role in livestock infectious diseases. is required in dogs. Immunization of cattle populations may be helpful in overall control of the disease. Standing is recognized.

EraSmuS, 5 1 Au5t Vet J 2 1 Ro (1975) .

It is therefore an object of the present invention to provide a vaccine with the above advantages, and , in particular provides an effective vaccine to protect cattle, sheep and sheep against BTV. That's true.

Special table Showa 58-50221υ (3) According to the invention, a group that is not of bovine, sheep, ovine or bovine wild mammal origin The bluetongue virus was adapted and grown in a cell culture from Oriyori. The pathogenicity has changed to such an extent that bluetongue virus disease cannot be recognized by injection. It was found that the virus was weakened.

In other words, the present invention relates to Bluetag virus serotype lT10. lT11, ITIro or one or more of lT17, at least about 104 pairs for T. Tissue culture containing only the infectious dose of IT10, ITII, ITI and IT17 blood To provide a vaccine for immunizing wild animals against clear bluetongue virus. here The virus serotype is determined by the pathological response of the disease caused by the Bluetag virus serotype. It stimulates the production of host defense mechanisms comparable to those that occur in natural infection, without causing infection. How is it weakened?

The present invention also provides a vaccine against bluetongue virus for injection administration. A manufacturing method is provided. In this method, bluetongue virus is cultured in Ver. .

Cell cultures are passed through bluetongue virus 60 times or more. This includes culturing and attenuating the bluetongue virus.

The invention also immunizes cattle, sheep or sheep against bluetongue virus. provide a method. With this method, each passage will take from 1 day to about 10 days. Ver. lT10 attenuated by passing it through cell culture 60 times or more , lT11, lT13 or lT17 bluetongue international virus serotypes. At least 2 cc of vaccine containing at least one or more than one species of or injection administration to sheep.

The present invention also provides for 6D times or more as described above in Vero cell culture. Brush germ-free embryonic chickens at least 6 times on the IJ rabbit before passing. Bluetongue virus of international type 16, weakened by passing through India. containing at least about 104 tissue culture infectious units per chain. Cattle, sheep, including administering CC vaccines to cattle, sheep or sheep by injection. Or to provide a method for immunizing sheep against bluetongue virus.

This vaccine is safe in that it does not cause any obvious disease in the animals receiving injections. Ru. Furthermore, the virus can be transferred from vaccinated fertile animals to other fertile animals that come into contact with them. There is no risk of pathogenicity returning by passing through the species. It is safe. The result is a significant improvement in BTV defense.

The basic method for producing the vaccine of the present invention is to Cultivate bluetongue virus by passing at least the O passage through the cell culture. This includes nurturing and attenuating. The Vero cell line will be explained in more detail later. As shown below, it is derived from African C) rreen MOn-key. . A series of passes was carried out at an incubation temperature of about 00C to about 60C. , lasting from about 1 to about 10 days.

The vaccine may be produced in a liquid form suitable for injection testing in veterinary animals. This liquid wax Cutin protects each BTV serotype it is intended to protect against at least about 104 Contains tissue culture infectious units. Dose suitable for immunizing cattle, sheep or sheep is approximately 2 cc of such liquid vaccine.

Live pathogenic bluetongue virus was isolated using isolation and identification methods described in the literature. virus, and can spread from infected cattle and sheep. Tobeba Livingst onand Moore, 25AJVR (1962)', Croldsmlt and Barzilai, 22. Refuah Vet 279 (196 5); See Luedke et al., OAJVR511 (1969).

The bluetongue virus serotype that is most useful in this invention originated in the United States. The International Serological Standards are 10.11.1 and 17. Of course, these The specific depiction of the virus is for illustrative purposes only and the present invention may not be applicable to other BTV serotypes. Needless to say, it is useful. These four types of viruses were collected on agar plate using the conventional method. They are serologically related (i.e., each virus is the same) as determined by the cell sedimentation test. type of antigen). However, 8 There is no cross-protection in sheep inoculation tests or conventional serum neutralization tests. These 4 The serotypes of the species were determined by De VillierS in 3 Intervirology. Among the several bluetongue virus serotypes described in 47 (1974) include. BTVITlQ, ITl　1, lT13 and lT17 serotypes are , U.S.A., Maryland, Rockville.

The American Type C11ection (ATCC) and the Catalog of 5tra1n Registered as VR-187, VR-872, VR-873 and VR-875 during recorded.

During the production of the vaccine of the invention, subculture levels 128 to 150 African Green Mon-key (Cercopithe NVero, a continuous cell line derived from For sensitive cell lines, low temperatures, preferably as low as 66°C, are most advantageous. For each BTV serotype, culture and propagate at 62°C continuously at least 0 times. (BTVITI filter is used for 6 blind passes during embryonic development.) can be first adapted to chicken eggs) to achieve attenuation and transformation of pathogenic BTV. sell. After every 5 passes out of 0 consecutive passes, the average pore size is 220 nm. The membranes were filtered and chloroformed before adding into the master pool, and the standard Virus can be purified by terminal dilution.

When starting the production of the vaccine of the present invention, USDA, 210(4) thropod born Viral Diseases Laborat ory, Denver.

using whole blood collected from infected sheep, such as that provided by Co1orado. Ru. The BTV strain with the lowest transit during the year can be used. Serotype BTV IT 10 , 11, and 17, washed red blood cells were destroyed and cultured in a monolayer culture of VerO cells. Adjacent to the nutrient layer, Dulbecco and Vogt + '99J, EXptlM ed 167 (1954) and Younger.

35 Proc Soc Exp Bj, ol and Med 202 (’ (1954) method and kept blind until an appropriate cytotoxic effect appears. Process the passage. Then, each of them used the wood end dilution scheme (・, 6 times Rack refine.

Attenuates the descendants of the black purified virus.

BTVITI (strain 67-41B), which is pathogenic to sheep, is a pathogen for the destruction of washed sheep red blood cells. 0.11n1. into chicken embryos by intravenously inoculating 12-day-old embryos with Adapt first. These embryos are collected after a certain period of time after inoculation, and the collected embryos are A 20% suspension of the heart tissue was passed through chicken embryos six times at the same inoculation volume. So Each passage lasts 6 to 7 days. Thereafter, this specific serotype BTVIT1 Adapted to vero, mB cylinder, standard DulbeCC, 0 method, Ro3 Proc Plaque purification is performed using Nat Acad Sci 747 (1952).

The time for passage is such that it allows maximum virus proliferation. normally, Maybe 1 to 10 days, preferably 2 to 7 days. The optimal transit time is standard 0 It can be determined by cell culture procedures. For example, a large man's cytotoxic effect (CPE) was observed. and the virus reproduces until 75 to 100% of the cell monolayer is destroyed. The time shall be .

Thus, the present invention provides a method for attenuating pathogenic strains of BTV. these stocks can be used alone or in optimal proportions and injected into sheep, sheep or cattle, preferably intramuscularly. injection to immunize with cognate BTV. In the method of the present invention, from about 0'c to about 6°C 1 Pathogenic BT in cell culture medium for 1 to 10 days, preferably at 62°C. V seeds are inoculated, cultured, and sequentially passed through additional such Ver○ cells. In total, it can be passed at least 600 times.

The virus preparation produced by the method of the present invention can be diluted to adjust the titer, and lactose, A stabilizer such as dextrose or other non-toxic substance may be added. virus style The product may also be dried, stored, and formulated into a liquid vaccine by methods such as lyophilization. Shitsuru. Stabilizers for virus lyophilization include Cryob, Rightsel, etc. iology42ro (1967) and Oreiff et al., Aduances 1 Described in nFreeze Drying, 10ro-122 (1966) .

Next, the present invention will be explained in more detail using specific examples.

These examples include the BTV vaccine of the invention, and methods for producing the vaccine and vaccine. A method for immunizing sterile animals using Chin is described. The latter two are both part of the present invention. Ru.

Example 1A BTV Texas 5ta, a live pathogenic bluetongue virus sample tion 5 train, IT 11 serotype (Hitonobin 2989, 7 days after infection, Whole blood in OPO) was obtained from 4°C-'C-stored whole blood. Blood 6. Qcc Centrifugation was performed at 1000 rpm for 10 minutes. Discard the supernatant and remove the clogged red blood cells. 1.0 mark balanced salt solution [phosphate buffered saline (PBS)] suspended. Cells were disrupted in a TenBroeCk tissue grinder, and the disrupted cells were P The final volume was filtered with BS and closed with QCr. 0.5 CC amount of disrupted red blood cell suspension Vero cell supernatant was added to the de-celled monolayer and the standard prepared as described below. Adsorption was carried out at 34° C. for 1 hour through the filter in a tube (16×125+==).

The VerO cell lines used were Yasamu, RA, and Chiba Oaza in Japan. It is a cell line started by Kawakita. 21 N1pp on R1n5ho 1201 (196ro), this cell line has passed 966 times. Laboratory of Tropical Viro from Chiba University Logy.

National In5 posture of Allergy and I nfeCtiOn, 5Diseases, Natj, onal In5tit Brought to you by the ute of Health. Then, this cell line passes American Type C1ture Co11 at level 113 Deposited in eclon. From ATCCK, CCL 81 VerO Panoseji It is distributed as a plate 122. Non-essential amino acids (NEAA) 12 1 part, fetal bovine serum 10 parts, sodium pyruvate 1% and kanamycin 200 parts Earle's Balanced Salto Solution (E M Ron) Medium Eag1. e’sMinimum Es5nential Medi Each cell line tube contains 2 cc of growth medium consisting of um. i to this ce suspension VerO cells (approximately 250,000 cells/CC,) were inoculated.

The pH was maintained at about 7.0 to 7.2 using sodium bicarbonate.

Cells were grown at 4.5°C for approximately 6 days to form a confluent monolayer.

Pour off the growth medium and place the cell monolayer in pre-warmed Hanks Balanced Saline. Red that was destroyed by 3.5cc of virus infection after washing with Letonorun Yong (Ron SS) Added blood cells. Adsorption was carried out at 60 to 64°C for 1 hour. Ro-kei horse face W, 2% Liptose Phosphate Pros, 100 units peningin and i　o　mc g streptomycin was supplemented. HBSS Medium Ba5a I Mediu, m 2 cc of maintenance medium consisting of Eagle (BME) was added. rollerdora The tubes were incubated at 60-64°C in a room incubator. 1 Approximately 6 to 5 such tubes were used for each round of virus passage. that's it Develop CPE until 75 or more cell monolayers are destroyed in passage. I let it happen. Samples were rapidly frozen at -85°C.

Collect the contents of the tube and repeat the passage (repeat for 30 to 50 additional passages). I let it pass. 29th special show! 18-5 (1221O(5) The 69th and 49th passes included additional passes to increase capacity.

A precursor to passing through to increase capacity is Feldman and Wang, 10. 6 Proc SOc, ExptlBiol Med, 7ro 6 (1961) Treatment of the virus suspension with chloroform and growth in antibiotic-free medium by the method of Ve A confluent monolayer of ro cells was inoculated. The maintenance medium in case 2 is 10% sheep serum. and BME (H) supplemented with 10% tryptose broth. At maximum CPE , the sol of the 40th and 50th passages of No. D1 was collected by a conventional method, identified and tight region.

The pool thus prepared was used as the bulk of the vaccine. This is in response to Quiter. diluted or added stabilizers or other non-toxic substances. Used as a vaccine (・ In order to make the product, dry it together with other BTV serotypes prepared in the same way. or can be prepared in liquid form as a multivalent vaccine.

When growing or attenuating viruses, use any non-toxic liquid tissue culture medium. It is also used. In addition to supplemented EM and BME media (described above), other Any non-toxic liquid tissue culture medium may be used.

Example 1B Example 1 was repeated, but BTV Texas 5tation 5train was replaced. Live bluetongue virus BTV BT 262, lT17 serotype (62 -45s, Sheep 279 [1, Infection 4 Seven days later, whole blood in OPC was used. This sample BTV BT262 is B of Example 1A. It was treated in the same way as the TV Texas 5tation 5train.

Example 1C Example 1A was repeated but replaced with BTV Texas 5tation 5train. In addition, live pathogenic bluetongue virus BTVBT3, lT10 serotype (sheep notification) 2617, 7 days post-infection, whole blood in OPG) was used. This sample is BTVBT8 (2617) is the BTV Texas 5tation 5train of Example 1A. processed in the same way.

Example 1D Example 1A was repeated but replaced with BTV Texas 5tation 5train. In addition, the pathogenic bluetongue virus BTV6741-B, ITl serotype (B TOX 190, Mutan 27ro1, 7 days after infection, OPG medium) were used. this Sample BTV6741-B is the BTV Texas 5tationStr of Example 1A. It was treated in the same manner as ain. However, immediately before inoculating the VerO cell culture. , a disrupted suspension of washed sheep red blood cells (o, ice) was injected intravenously into 12-day-old embryos, and then into chicken embryos. adapted. Embryos were collected at intervals after the initial inoculation.

The heart tissue of the collected embryos was made into a 20-fold suspension and transferred to chicken embryos at the same inoculum amount. did. The time for each passage was 6 to 7 days. Adapted to chicken embryos and samples were adapted to Vero cells as described in Example 18.

Example 2 Example 1 Mark 2 of the vaccine (xTll) manufactured by AK ) passed 20 times and measured with CPE from 60 to 105°0TCID at 4°C. It was titrated to 5o/CC and injected intramuscularly into five one-year-old lambs.

(TCID5□ - tissue culture infection dose giving 50% CPE at highest dilution). other Five sheep served as unvaccinated controls. These 10 sheep are all BTV It was confirmed that the serum was negative. Serious (・Evidence of BTV disease is It usually manifests as a fever 5 to 8 days after exposure to primary BTV and other symptoms. Bed symptoms also appeared on days 6 to 8.

Antibody strains from five inoculated one-year-old sheep were tested by agar gel precipitation (ALP) test. Test results were negative. The score was 15 or less on the direct emotion neutralization test (SN). 1 month later, 5 heads The AOP test was positive for antibody typhus for the sheep, and 6 sheep ( The N test was more than 120 dogs. In no case were both tests negative. 1 of 10 For all age sheep, 500-1000 sheep infectious dose (SXD)/CC, (sheep 2 cc of the homologous pathogenic virus containing 2 cc of the homologous pathogenic virus was inoculated. sick Symptoms were observed for several days. All five vaccinated sheep were normal and showed no clinical signs. There was no. The five unvaccinated animals contracted BTV disease, exhibiting fever, loss of appetite, and general symptoms. The patient showed severe discomfort, C0rOnitiS, and ulceration of the gums.

16 For example, A A live pathogen isolated by standard tissue culture techniques and designated as BTV IT, 11 BTV of the same sex were plated onto continuous VerO cell monolayers at intervals of 6-7 days at approximately 62°C. This was followed by 10 consecutive passes. 1c passes tested on sheep Then, a febrile reaction occurs and mild clinical signs of TV infection appear. Considered to remain pathogenic. Whole blood collected at the peak of infection can be added to Passed through the passive sheep. And then I looked at the clinical signs of the disease. Viruses are pathogenic there were.

Vero monolayer cell cultures were passed an additional nine times at intervals of 7 to 10 days. 19th time The passage was carried out to increase capacity. Virus about what was posted 20 times The content was determined by titerinone, identified by serum neutralization, and used in the test vaccine. 20 times Vaccination of sheep with the pass-through gives significant amounts of antibodies, making the sheep resistant to pathogenic BTV. became resistant. However, this 20-passage product has residual pathogenicity, judging from the exothermic reaction. showed that. Other clinical signs were isolated using standard tissue culture methods and BTVITl A live pathogenic BTV, designated as BTVITll, was described in ℃TereroA. I passed it in the same way. The same series of passes was performed with the sheep, BTVITll of Example 3A. got the same result.

Example 6C Live pathogenic BT cultured and isolated using standard tissue culture methods and designated as BTVITl. V was passed in the same manner as described in Example 3A for BTVITll. sheep , I passed it in the same way using the same method, and got the same result as BTVITll in A. Ta.

Example 4A After 20 passes of filter A, the virus material was passed through filter O at 340C to confine Vero cells. A number of passes were made through the tube containing the luent monolayer.

After the 29th and 69th pass, the virus progeny spreads further and passes through the 60th and 4th pass. The material was made to pass 0 times. Each pool was produced in antibiotic medium and had approximately 10ξ TCID,. /CC, (measured by CPE) The cells were frozen at 85°C. Ver○ The virus before inoculating into the cell monolayer is chlorophotized. This treatment inactivated the contaminants and increased the infectivity of the virus.

The 30 and 40 passes of the sol were used in vaccines in the manner described in Example 2.

Briefly, five susceptible sheep were injected with 2.0 cc of 60-pass material. vaccine treatment. The remaining five animals served as unvaccinated controls. ten sheep A preliminary test revealed that the serum was negative for BTV. Each year, each year is about fever. The animals were monitored daily to see if there were any clinical symptoms. After 28 days, for all 10 animals, approx. Contains 00 to 1000 S I D/CC8 Total dose 2. The virus was inoculated with OCc. Regarding the presence or absence of clinical signs Observations were made for 4 weeks. The five vaccinated sheep were normal and showed no clinical disease or disease symptoms. won. The five unvaccinated animals became sick with BTV and the amount of virus inoculated was Typical symptoms were fever, decreased appetite, and general weakness. 40 times passed material I also looked into the fees. The results were similar to those observed for the 60-pass material. Ta.

Example 4B The 20-pass virus material of Example 3B was passed in the same manner as Example 4A. BTV IT Ten 0-pass and 40-pass materials were obtained.

Example 4C The 20-pass material from Example C was passed through BTVITlo as in Example 4A. The material was passed twice and 40 times. Pooling these 60 and 40 times materials, e.g. As in 4A, BTVITll was tested with similar results.

Example 4D The 20 passes material from Example 2 was passed as in Example 4A and 0 passes of BTVITlo. and 40 passes material was obtained. These 0- and 40-pass material barbs were prepared in Example IV. It was tested in the same manner as BTVITll of A and the same results were obtained.

Example 5 Two serotypes, IT 10 and IT11, IT11 and IT17, and Examples 1A to 1c of viral materials containing combinations of T10 and lT17 It was prepared by the procedure detailed in . of these serotype combinations at the 50-pass level. Pour 20Ω amount of each material into a standard vaccine bottle and heat at -90°C. The cells were frozen at -80°C and stored until inoculation. Freeze bottles quickly Thaw and apply approximately 10” TCI to each of the 6 sheep (2 used for each combination). A titer of D5o/, cr, was injected for 2.0 CC. Sheep are housed separately The patients were checked daily to see if they had developed any symptoms. Blood was collected on days 7 and 14 after vaccination. They were collected in OPG and virus isolated. All sheep are treated with sushi before vaccination. I drew blood and checked for antibodies.

After 1'r months, vaccinated sheep receive 500-100 of the serotypes represented. Of the two dogs, one of them wanted to be inoculated with the pathogenic virus developed by SID/CC. (using a different virus). The sheep were observed daily until 4 weeks after vaccination, but no clinical symptoms were observed. There wasn't. In contrast, unvaccinated sheep developed typical BTV disease.

After 2 months of vaccine treatment, a dose of 500-1000 SIDA will reduce the pathogenic virus. Inoculate the sheep with another virus that was used previously.After inoculation, the sheep were was observed, but there were no signs of illness. In contrast, unvaccinated control sheep The patient showed typical onset of T V disease. Antibody measurements before and after vaccination , before vaccination, all sheep had negative serum in the agar gel sedimentation test; After treatment, 4 out of 6 animals had positive serology. 7th and 1st molar after vaccination A blood sample collected on day 4 tested negative for circulating vaccine samples.

Vaccinated sheep were completely protected from disease compared to unvaccinated controls.

This example shows that the attenuated bluetongue virus contained in vaccines is not fully protective. This indicates that the titer is not high enough to be detected by conventional laboratory methods.

Example 6 The 15-pass level vaccine was administered to four serotypes as shown in Examples 1A to 1D. were prepared for each. Vaccines are placed undiluted in standard vaccine bottles. It was frozen at -90"C to -80°C and used for inoculation. The cutin thawed quickly.

Each of the 20 sheep has 2. OCr- was injected. Blood was collected from all sheep, Check the antibody titer before vaccination and check for fever or side effects to the vaccine. I watched it every day. One month later, each of the five sheep had a different serotype of pathogenic BT virus. was inoculated. In other words, 5 dogs have BTVITiQ, 5 dogs have IT1.5 dogs have IT. 1, 5 of them are 1T17. Unvaccinated controls were also inoculated with the virus. did.

One serotype, ■T10, failed to protect against the pathogenic virus. 6 other stocks defended. All serum samples were negative for antibodies. Check out the remaining vaccines If you look, the vaccine type for Engineering T10 is 102T CI D50/CC U. It was less than a virus. In this example, the vaccine did not work due to processing difficulties. Show that.

Adequate protection will have been achieved in vaccinated sheep if sufficient amounts of virus are present. Dew.

Example 7A The vaccine was prepared as in Example 1A, but using a 50-dose bell. Waku Chin is 104TCID5o/σ, 10"TCID/cc or 102TC 0 containing I D5o /cc diluted as follows. Then, put it in a standard vaccine bottle and use it for temperatures ranging from -90°C to -90°C. The samples were frozen at 80°C and used for inoculation. Frozen bottles thaw rapidly. 72 sheep to understand 2. I gave an injection to OCC. 24 animals are 104TCID5o/C 24 Cl heads are 103TCTD1. . /LL and 24 heads are 102TCID5o/ A CC Quiter was used. All sheep were bled and tested for antibody production before vaccination. I saw it.

One month later, all vaccinated sheep were bled and their post-vaccinated cuiku was examined. A pathogenic virus of 500 to 1000 SID/Cm of the family virus was inoculated.

The sheep were observed daily until 4 weeks after inoculation, but no disease symptoms were observed. However, each There was an increase in body temperature in the group. The exothermic reaction was 104TCID5o/CC and unvaccinated. It was weaker in the Chin-treated group. Measuring antibodies before and after vaccination shows that the agar In the sedimentation test and serum neutralization test (serum dilution 1.5), the serum reaction against the vaccine was The test result was negative.

However, vaccine-treated sheep were completely protected from the disease.

This example shows that although the vaccine is protective, the antigen is Repeat Example 7A, but instead of BTVITII, 50 times of BTV T10 - Use the bell at ℃・ro. This sample (IT10) is the same 103TCID5o/CC1102TCID5o/Cr- and 101T CI D50/CC is used.

Similar results were obtained with 103TCID5o/CC. The other two quantities were ineffective.

Example 7C Do the same as Example 7A or replace BTV IT 11 with BTVIT17 50 times A call bell was used. This sample (IT17) was treated similarly. However, 105T CID5o/CC1104TCID5o/CC and 103TCID5o/CC was used.

Results similar to Example 7A were obtained.

Example 8 By the procedures described in Examples 1A to 1D, four serotypes BTVIT10, IT11, IT Viral materials containing 13 and lT17 were prepared. All 4 serotypes Contains all of this viral material containing 50 doses of Put 2,000 cc amount into a standard vaccine bottle, minus 90 to minus 80 The samples were frozen and stored at ℃ and used for inoculation purposes. Freeze bottles quickly 5o/Cr- from about io5°5TC to each of 1000 sheep. 2. with Quicoo. I gave an injection to OCC.

The sheep were kept in groups: 11 A1 groups 500, 3 groups 250, the rest were C and and group D. Randomly select 50 sheep from groups A and B, collect their blood, and administer the vaccine. I looked at the antibody quirk before treatment. Then I marked it and read it further. C and D Blood was drawn from 10% of the b+ samples in advance and marked in the same manner.

After one month, the marked vaccinated sheep were each given 500-1000 SID/Cm. Pathogenic viruses consisting of four types of blood type M were inoculated. Observe sheep daily for 4 weeks after vaccination However, there were no symptoms of illness. In contrast, vaccinated sheep had BTVfp A typical fishing machine is shown below. Antibodies before and after vaccine treatment were tested by agar sedimentation test and A serum neutralization test (serum dilution of 15) was performed, but the serum was negative. However, the vaccine Treated sheep were fully protected compared to unvaccinated controls.

This example shows that the attenuated bluetongue virus is completely protective, but The serum test shows that the antigen marker has been lost and it shows a ℃ filtrate. The function of this example The problem was repeated twice, one year apart, with similar results.

Example 9 The vaccine was prepared as in Example 8. However, 40 times 24 Excessive levels of materials were used. Approximately 105” Virus tie of 50/CC from TC 2Cr- vaccine for susceptible cows (5 head) and sheep (100 head) was administered by injection. They were seronegative for BTV. another cow and sheep served as an unvaccinated control. What is the overall antibody status of the animal before vaccination? Less than 1.5 for sheep and less than 1:10 for cattle (serum neutralization test) and agar gel Sedimentation test was negative. One month later, the antibody titer just before virus inoculation was The Kell sedimentation test went from negative to positive, and the serum neutralization test went from 1:20 to 1.4o. It changed to All animals were injected with pathogenic viruses of all four serotypes. gave. 500-1000 SID/CC for each serotype, total dose 2.0 It was set as CC.

The presence or absence of fishing equipment for cattle and sheep was observed for 30 to 90 days. Vaccinated animals are positive As usual, fishing machines were not allowed at all.

Example 10 200 cc of each of the 40-pass viruses obtained as described in Examples 4A-4D. samples were used for inoculation. Before vaccination, all 1-year-olds were tested for serum neutralization. The antibody titer was less than 15, and the agar gel sedimentation test was negative. Storage of each serotype The mother pool of BTVIT was diluted to a content of 5o/LOf- from about 10TC. 10.11.1 Multivalent work containing 10 and 17 equal types Special Law 58-5022111 (8) The remaining mice were left untreated with the vaccine and served as controls. Each year, The animals were observed daily for fever and vaccine side effects. One month later, 20 animals were vaccinated. The sheep were divided into 4 groups of 5 sheep each, and each group had one BTVITlo and 11.1 sheep. and 17 inoculated with 500-1000 SID/CC of pathogenic virus. So Appropriate unvaccinated controls for each group were also provided.

Sera were collected immediately before inoculation with the pathogenic virus and examined by an agar gel sedimentation test. Wakuchi More than 60% of the treated group showed the presence of specific BTV antibodies in their serum. However, I'm excited There was no antibody in the untreated control. All 20 sheep treated with vaccine were given 3 The cells were inoculated with another BTV serotype virus at an interval of 0 days. And fishing machine every day saw.

The scheme of virus inoculation is shown in the table. Vaccine-treated sheep do not show signs of fishing, and vaccine Untreated sheep developed BTV disease.

Typical signs: fever, rash, lethargy, loss of appetite, and general discomfort showed that.

A 30th day 60 90 120 B 120th day Ro0 60 90 C90th day 121] 30 60 D 60th day 90 120 50 Each group was inoculated with the indicated serotype on the indicated day. Appropriate controls were provided for each inoculation. .

Example 11 A 20Qcc sample of the vaccine produced in Example 10 was used for inoculation. Before vaccine treatment, fruit Eighty percent of the trials were negative in the agar gel sedimentation test. of stored viruses of each serotype. Dilute the mother pool to about 104T CI D50/cc and make BTV Ti Q.2 of a multivalent vaccine containing equal titers of 11.1 and 17 cc was injected into each of 69 pregnant (terminal) cows. Other unvaccinated A control was provided. After 50 days, blood was collected from the cow and subjected to serum precipitated antibody analysis. I checked to see if there was a virus.

Furthermore, after 96 days, 58 calves born from cows and vaccinated calves were also examined. Examined.

All dish fluid samples were negative for the virus, and all vaccinated animals were normal and showed fishing equipment. I didn't. The product was safe, non-abortive and non-teratogenic in terms of newborn calves. . Furthermore, 41% of the calves were positive for antibodies in the agar gel sedimentation test, indicating that maternal antibodies were detected by ingesting colostrum. Indicates physical acquisition.

Example 12 500 CC of the vaccine prepared in Example 10 was used for inoculation.

All cows were palpated for pregnancy prior to vaccination. 58% are pregnant I was pregnant. Dilute the stock virus mother pool of each serotype to approximately 104 TCID. BTV IT 10.11.1 and 17 prepared as 5o/CC 2CC of polyvalent vaccine containing Iku equivalent from 45th to January 8th. Each of 180 cows at different stages of pregnancy and 4 bulls were dosed.

Cows were observed daily for clinical disease and abortion. After 60 days, cow pregnancy The patient was palpated for the presence or absence of Person in charge 72 was pregnant. This means that vaccines cause miscarriages. However, as shown in Example 10, manufacturing defects may not cause temporary or long-term infertility. A 53 cc sample of Chin was used for inoculation purposes. All fruit should be cleaned before vaccine processing. There was no circulating virus in the Shiraoko used for testing.

Eight of the 12 cows developed agar gel precipitated antibodies (antibodies from the mother) by ingesting colostrum. had. The stock virus mother pool of each serotype is approximately 104T CI D5 Equal to BTVITlo, 11.1 and 17, obtained by diluting to 0/cc A 2 cc dose of the multivalent vaccine containing Cuicu equivalent was administered to each of the 12 Shira-tailed calves. The patient was given an injection. For each measles, presence or absence of fever and other side effects caused by the vaccine. I saw it. 4 and 9 days after vaccination, blood was collected from each cub and the vaccine. I saw the presence of Chin virus.

One month later, two of the vaccinated pups were infected with pathogenic bugs of 250-5008 ID/cc. inoculated with the virus. Each measles contains BTVITlo, 11.1lo and 17 Ta. Others were voiced in opposition. Blood was collected from each deer 8 and 15 days after inoculation with the pathogenic virus. and confirmed the presence of pathogenic viruses.

28 Low levels of BT■■T10 are present in the blood of the baby's head 9 days after vaccination. did. No other serotypes of vaccine virus were isolated. More pathogenic viruses Blood samples from two animals 8 days after inoculation contained pathogenic virus. 1 head+! B Tv IT 10 Te, 1 head BTV I T17. After vaccination Neither the deer nor the deer showed any signs of fishing after virus inoculation. This means that the vaccine The attenuated bluetongue virus contained in the bottle has a completely protective effect, but it is not It always shows multiplication scales in young susceptible animals.

Based on the above specific examples, each of the bluetongue viruses must meet the criteria. It turns out that this is attenuated enough to be used in vaccines.

(1) A small amount of attenuated virus (approximately 104 tissue culture infectious doses) More than 70% of cattle, sheep and sheep injected with the vaccine containing After 4 hours, 500-100 D SID/CC-containing viruses were detected. Infect cows, sheep, and sheep with 2Cf by injection; they must survive even after vaccination. That's not true.

and (2) Pregnant cows, sheep and ewe at the Zenzen pregnancy stage, 10 to 10 TC Receiving injection administration of 2CIC of vaccine containing ID50/cc. Even if you get pregnant, you should not get sick and have a miscarriage.

Special table Showa 58-50221fJ (9) Procedural amendment (formality) %formula% 1.Display of the incident PQT/σs 83100052 2. Name of the invention Bluetongue virus vaccine, its production method, and anti-separation behavior caused by this vaccine How to immunize things 3, person who corrects Relationship to the incident: Patent applicant full name (Name) → Country ``47'' ``4 = ° Par'ν Putty 45, date of correction order October 11, 1982 6. Number of inventions increased by amendment international search report Continuation of page 1 0 shots Mings Josia A Texas 77843 United States College Station, Texas (no street address) Texas A&M Uni Varsity College of Veterinary Medicine Department Veterinary microbiology and biological awareness 0 shots clearer livingston charles w jr.

Claims (1)

[Claims] (1) When the vaccine was administered parenterally to wild animals, bluetongue virus serotype host disease comparable to that produced by natural infection, without exhibiting pathological morbidity responses caused by Bluetongue virus serum attenuated to 5, which can stimulate the production of resistance mechanisms. Type lT10. one or more of IT11, IT17 or IT17 Bluetan containing at least about 104 tissue culture infectious doses of Virus international serotypes IT10, T11, ITiro and IT1'7 , a vaccine to immunize against animals (2) At an incubation temperature of about OoC to about 6°C, each pass is about VerO cell cultures in culture 30 times or more, lasting from 1 day to about 10 days. By passing more than one bluetongue, one or more bluetongues can be The vaccine according to item (1) above, which attenuates each of the international virus serotypes. (3) Pathogens that are becoming embryonic before passing through Vero cells 60 times or more Attenuated bacteria-free chicken eggs passed through at least a rotor blind; Approximately 10' tissue culture infection dose of bluetongue virus international serotype 1C The vaccine according to item (2) above, containing per C. (4) Where the vaccine is in a dry solid unit dosage form and is dried at low temperatures to become a solid ( 2) or the vaccine described in (3). (5) The time required for passage during Vero cell culture is approximately 2 days to approximately 7 days. The vaccine according to any one of the above items (2), (3), or (4). (6) The animal is selected from cattle, sheep, or sheep, and the vaccine is Contains about 104 to about 106 tissue culture infectious doses of virus serotypes per omega. The vaccine according to any one of items (2) to (5) above. (7) Zero or more passes through the Vero cell culture in culture medium Injecting into sterile animals, including culturing and attenuating the Plutoung virus. A method for producing a vaccine against bluetongue virus. (8) Bluetongue virus international serotype IT 10°IT' 11, IT 1 At least 104 tissue cultures of 17 or more IT The method according to item (7) above, wherein there is a nurturing dose. (9) The virus is Bluetongue International Serotype 13 (BTVITIro), (1) At least 6 times, BTVIT13 was applied to rabbits during pathogen-free embryogenesis. Cultivate the virus, weaken it, and (2) Pass 0 or more times through the Ver○ cell culture in the culture medium. The BTVIT virus was cultivated and attenuated, Bluetongue virus international serotype 13 (BTV IT 13) 31 A method for manufacturing vaccines used for injecting for protection. α1 At a temperature of about 0℃ to about 66℃, each pass takes about 1 day to about 10 days. The virus was cultured and attenuated by passing it into a Ver○ cell culture. The method according to item (7), (8) or (9) above. qI) (7), (8), (9) or 00 above, where each passage is from about 2 to about 7 days. The method described in any of the paragraphs. (6) from about 30°C to about 36°C, with each passage lasting from about 1 day to about 10 days; Filter the virus into Vero cell cultures in culture medium at incubation temperature. Bluetongue international serotype IT 10, l, which has been attenuated by passing or more At least one or more than one of Ti1, IT13 or IT17 At least 2 cx of the virus-containing vaccine are injected into cattle, sheep or sheep. Infection of cattle, sheep or sheep against bluetongue virus, including injecting How to get discharged. (13 Before passing the chicken into the Vero cell culture 60 times or more) Attenuated by at least 6 blind passages into embryonic pathogen-free embryonating eggs. , at least about 10' of bluetongue virus international serotype 13. The immunization according to item 4 above, which comprises administering the dose to cattle, sheep or sheep by injection. Method. 1*a　1982-502210　(2) Engraving (Contents (no changes))