HU187793B - Process for producing attenuated living virus vaccine against dog-distemper - Google Patents

Abstract

The present invention relates to a method for producing an attenuated live virus vaccine against sorghum. According to the invention, the modified Lederle paramyxo virus ML-32 deposited in the National Collection of Microorganisms of the National Institute of Public Health is cultured for 25-40 passages on primary or secondary fibroblast cultures, the virus fluid thus obtained is described as a vaccine; the vaccine is inoculated with further primary or secondary fibroblast tissue cultures, incubated for 4-9 days, and the virus fluid is isolated and lyophilized after the addition of a protective colloid. The vaccine prepared as described above is capable of active immunization against dogs of 3 months of age or older. 4 s Y • J 5 ΐ s 1 ϊ 1 -1-

Description

The present invention relates to a process for the production of a live viral vaccine for attenuated virulence in dogs in cell culture.

Birdworm virus has been known in Europe since the second half of the 18th century. Some say the virus originates in Asia, others believe it comes from Peru. Its infectious nature has been known since the middle of the last century, and Carré (1905) recognized the viral origin of the disease called febris catarrhalis infectiosa canunt.

Piglet is a very common infectious disease in dogs. Almost every dog goes through it. It is most common at the age of 1-2. The disease is especially widespread in the summer, as this is the season when dogs are most likely to become infected. In summer, the disease is usually benign. In contrast, in winter and spring, it often occurs in a complicating form. In addition to the dog, wolves and foxes are extremely susceptible to the virus. Silverstroke often causes great losses in cultures. Glanders in fur breeding establishments (weasel, hermelin, mink, ferret) and in zoos also occur among otters.

Buckwheat Virus (Carré, 1905) belongs to the paramyxo group. The earlier assumption that the virus would be present in multiple immunobiological variants has not been substantiated. There are immunobiologically related relationships between bovine, oriental beef and human measles virus.

The infection, the nasal and lung secretions of diseased animals, is caused by substances contaminated with urine and urine. Susceptible olian dogs that have not yet died of the disease, with the exception of immune mothers younger than 6 weeks, who have acquired passive immunity by uptake of swallow milk. There is no need for predisposing agents to cause infection. However, certain predisposing conditions, such as colds, inadequate or inadequate nutrition, lack of exercise, inbreeding, helminths, and other maladies, occasionally play a significant role in aggravating the course of the larynx and contributing to the development of complications.

The first stage of the cilia is a pure incest, when the virus multiplies first in the lymphatic vessels, then in the lymphoreticular tissue, and then causes viraemia and, accordingly, fever. Highly susceptible animals are already victims of this infection, but usually the infection itself does not kill the patient. The temperature rise is temporary because it only lasts for a few days while the incest persists. Once the incest has ceased, the temperature returns to its original value and the virus heals when the virus is removed from the blood. However, in some cases, the disease does not end because the temperature rises soon, indicating that the circulating virus causes cataracts in each mucosa by loosening the endothelium of the blood vessels, and in some organs develops a profound disease. caused by the virus, partly due to the proliferation of bacteria that subsequently penetrated. Depending on the organs in which inflammatory phenomena develop, it has been customary to speak of cataract, nervous, and rash dust. Often, certain organ systems are at the forefront of the disease, but in general it is not possible to distinguish sharply the mentioned forms of the piglet, and in many cases the disease changes its nature over time. In almost all cases of suckling, the cataract appears early in the airway. In rare cases, the cornea also becomes cloudy or corneal ulcers are formed. Gastrointestinal disease is characterized by more or less severe throat inflammation accompanied by swelling of the tonsils and acute gastrointestinal or gastrointestinal inflammation. Symptoms of nervous system malignancy are also common in mild cases but sometimes severe. Nervous system symptoms include dullness or agitation, convulsions in some muscle groups, or occasionally recurring stiffness, paralysis. In some dogs, the skin of the abdomen and the hind limbs develops a special rash in some cases. Mild eczema on the external auditory canal is also frequently seen. Pregnant animals can miscarry. In some cases, the disease is complemented by a disease of the soles of the feet, previously considered a separate disease. In this case, the horn layer on the nose and especially on the soles thickens and hardens.

Buckwheat is a significant lesion in the development of the disease described. On average, 50% of the affected animals die without proper treatment.

Animals at risk of infection should be protected by active immunization. Nowadays, the most commonly used immunization is an attenuated, modified live virus vaccine in egg or tissue culture and lyophilized. At present, live viruses of weakened virulence in eggs are used in vaccine production in Hungary and in many countries. These viruses are mostly isolates of that country. The production of egg boar vaccines is extremely complicated and labor-intensive. Since the spread of avianized chickpea virus strains, many researchers have dealt with the propagation of different virulent or chick embryo virus strains in tissue culture. Many unsuccessful attempts (Cabasso, Accompany and Stebbins; Proc. Soc. Exp. Bioi. 1959-1005. 551 - Hopper; J. comp. Path. 1959;

P. K. 78. - Bussel and Kazon; Arch. ges. Virusforsch. Dedié and Klapötke after 17.1.1965; Vet. Med., 13 March 1959; and Rockborn; Arch. ges. Virusforsch. 8.8.1958 485 successful breeding virus of porcupine virus! report. However, the virulent viruses so propagated cannot be used to produce a live virus vaccine. Further, several researchers have attempted to propagate different strains of porcupine virus in chick embryo-fibroblast cell culture. The virus adhered to the tissue but did not appear to damage the cells of the culture (Reculard et al; Ann. Inst. Pasteur, 1960, 98, 344 - Cabasso, Kiser, &Stebbins; Am. J. Vess., 1962, 23,394). Jiran, Vet. Med.Praha, October 10, 1965 - Dias Vigario and Ferreira; Bohn. Pecuar, 33. No. 2. 131. 1965. Karzon and Bussel (Science, 1959. 130.1708) propagated the strain Onderstepoort. successfully in chick embryo fibroblast tissue culture. Exam-23

187,793

In their cells, viral replication was accompanied by a specific cytopathogenic effect. In their later studies, Busscl and Karzon found that tissue-trained Onderstepoort strains were later less able to induce lesions on the chorioallantoic membrane. Paganini and Papparella; Acta Med. Vet. Napoli, 6 June 253, 1960, with similar results, propagated avianized porcine virus strain in chick embryo fibroblast cell culture. Vasic. B., Vasic N. Lukic and Kristic (1976) also propagated the Onderstepoort strain in chicken embryo fibroblast cell culture. In our studies, virus growth was not followed by a cytopathogenic effect, and viral growth was confirmed by reversal on the chorioallantoic membrane.

There is contradictory literature on the growth of sturgeon virus strains in cell culture. In some cases, virulent strains of porcine viruses have been successfully propagated in cell culture, and in some cases, cell proliferation has been associated with viral proliferation. Among the strains of virus used for the production of vaccine against porcupine, the Onderstepoort virus strain was successfully propagated by tissue culture. In some cases, viral replication was accompanied by a specific cytopathogenic effect, but the virus thus propagated was later less able to induce lesions on the chorioallantoic membrane. Others have suggested that the strain of Onderstepoort virus grown in cell culture has no cytopathogenic effect and that viral growth can only be demonstrated by inoculation into the choriallantoic membrane. Virulent piglet viruses propagated in cell culture cannot be used for the production of live virus vaccine due to the spread or survival of the disease. Cell culture strains of viruses used for vaccine production generally have no cellular damage and viral growth and virus titer control can only be accomplished using sophisticated and laborious methods (such as the immunofluorescence method for the Rockborm strain).

It is an object of the present invention to provide a simple process for the preparation of a modified live virus vaccine which is propagated in cell culture and provides protection against porcine.

In our study, we used a strain of Lederle piglet virus adapted to the chicken embryo provided by Behringwerke A.G. The virus multiplied on the chorioallantoic membrane of chicken embryo and showed no cytopathogenic effect in cell culture. During our experiments, II-day germ embryo booby; fibroblast cultures were prepared by trypsin digestion. 0.5% lactalbumin hydrolyzate, 10% chicken blood serum or 10% calf serum, 100 U / ml penicillin, SOpg / ml streptomycin were used for culture. Prior to use, the culture medium was drained and an equal amount of a mixture of Hanks' solution containing 1% chicken and 1% calf serum and amniotic fluid was used as a maintenance fluid. The avianized Lederle virus was passaged weekly on the described fibroblast culture. The culture vessels were maintained at 37 ° C and examined daily. On day 7 after virus inoculation, the culture fluid was sterile aspirated and the harvested fluid inoculated. No cytopathogenic separations were found in the first 4 passages. 5.

at passage, the cells of the chicken serum culture shrunk in foci on day 4. On day 5, the culture began to rupture around the foci and by day 6-7. by day it became networked. The control cultures, however, remained intact. This tissue damaging effect of the virus can be consistently observed in further passages. Histological examination of 5-day cultures revealed multinucleated giant cells, while 6, -7. day cultures were characterized by a synaptic, networked structure and a decrease in giant cell numbers. In calf serum cultures, cell damage was only observed at passage 10. To verify the origin of the cellular damage from Porcupine Virus, a viral neutralization assay was performed on chorioallantoic membrane and tissue culture. Passage 10 was neutralized with 10 ^3.33 1DSO and 10 ^2.25 TCIDS0 titers against 1: 10 diluted piglet serum. In passage 10, the virus harvested on day 7 was centrifuged and lyophilized by the addition of an equal volume of 3 parts of horse serum, 1 part of broth and 7.5% glucose in a protective colloid. További In further passages of the virus, the medium necessary for the production of the scabies and the protective colloid for lyophilization of the virus were modified in both components and proportions to increase or protect the virus titer.

In the method of the invention, passage 32 of Lederle avianized strain of avian strain virus cultured in cell culture is used for vaccine production. Cell cultures suitable for virus propagation (primary or secondary chicken embryo fibroblast) were prepared using media containing 10% tryptose phosphate broth, 4% calf serum and 86% Parker 199. A tissue stock virus stock of a minimum of 10 ³ TCID _SO / 0.2 ml is stored at -196 ° C or lyophilized at -20 ° C. Tissue cultures prepared for vaccine production are inoculated with passage 1 of tissue inoculum virus stock in cell culture and lyophilized after the incubation period with the addition of appropriate protective colloid. The vaccine thus prepared is suitable for the active immunization of piglets of 3 months or older against porcine animals.

The virus strain used in the method of the present invention has a cytopathogenic effect, and therefore viral growth in cell culture can be monitored by light microscopy. The virus retained good antigen activity during serial passages and did not lose pathogenicity to the chorioallantoic membrane of the chicken embryo. Viral measurement can thus be performed both in cell culture and by inoculation onto a chorioallantoic membrane. For many other strain of cellulose virus grown in cell culture, virus titration can only be accomplished by a complicated and costly immunofluorescence procedure or only by inoculation onto a chorioallantoic membrane. The virus strain is propagated on chicken embryo fibroblast cell culture, which can be produced easily and routinely in any tissue culture laboratory. In many places, porcupine viruses are propagated in primary cultures of weasel or ferret cells, while elsewhere they are propagated on Blood cells. In many countries, including Hungary, is a multiplier3

A vaccine against 187,793 ducks is produced by inoculating or processing the chorioallantoic membrane of chicken embryos. This method is extremely costly and labor intensive. For the production of 30 to 40 thousand doses of the vaccine, the above method requires about 100,000 pieces of Einbri eggs. Approximately 150 to 200 embryonated eggs are required to produce the same dose of vaccine using a strain of chicken embryo fibroblasts used in the method of the invention and propagated under the influence of CP. 1000 For producing a vaccine on a chorioallantoic membrane, it will take about 100 hours to produce 1000 doses. In chicken embryo fibroblast cell culture, using the virus of the present invention, it is sufficient to produce 1000 doses of vaccine for about ten hours.

The invention is illustrated by the following example.

Example

Description of the cell-borne porcine vaccine (Distevac):

The virus is propagated on a primary or secondary fibroblast culture of 10-11 day old chicken embryos. Cultures are prepared by trypsin digestion of 0.07-0.08%. Cells were grown in 10% tryptose phosphate broth, 4% calf serum and 86% Parker 199. Cell culture overgrown in Roux flasks for 24-48 hours is inoculated with 15 ml of at least 10 ³ TCID _SO / 0.2 ml Titer virus (passage 32 in Lederle avianized pig strain virus cell culture). After one hour of adsorption, the amount of virus was replaced with 120-200 ml of a solution containing 10% tryptose phosphate broth, 2% calf serum and 88% Parker 199. Keeping the bottles at 37 ° C is the harvest

5th-6th days when the cultures show 80-100% cytopathogenic activity. The sterility of the collected viral fluid is shown in Table VI. Test according to Hungarian Pharmacopoeia. Sterile and at least 10 ³ TC1D _SO- titer-vaccinia virus stock is stored in plastic ampoules at -196 ° C and lyophilized at -20 ° C.

The vaccine is produced in the same manner as the production of the tissue inoculum virus stock. The collected viral fluid was lyophilized by addition of 1% gelatin, 1% sucrose, 0.75% tryptone and 0.5% collidone.

The sterility test of the lyophilized vaccine is described in Annex VI. Follow the instructions of the Hungarian Pharmacopoeia.

The vaccine must not contain live germ.

The viral content of the lyophilized vaccine was determined by titration on the chorioallantoic membrane of 7 days of pre-incubated chicken embryo or by measurement of viral value in chicken embryo fibroblast cell culture. Suitable vaccine, at least 10 ^ls ID _N5 / 0.2 ml vírustartalomma! It has. Λ The vaccine can be stored for 6 months at successful trial at -20 ° C. The expiration date of the vaccine in case of another successful test is the titer! 3-6 months, depending on the extension.

The safety and efficacy of the vaccine should also be randomly tested in animal models. The vaccine administered at a practical dose should be used to inject a 3 '1-month-old dog susceptible to 3 piglets into the thigh muscle. Inoculated dogs should show no signs of disease during the 21-day observation period. On day 21 of the study, vaccinated dogs, preferably with 2 unvaccinated controls from the same litter, received 10,100 LD virulent piglets

Comes with 2g virus! infect intracerebrally. During the 14-day observation period, vaccinated dogs may become mildly affected only temporarily. One of the two control animals should die in the littermate by the end of the observation period.

Claims (2)

Patent claims A method for producing an attenuated live viral vaccine against porcine animals in cell culture, characterized in that ML-32 modified Lederle's paramyxo virus modified by depositing ML-32 in the National Collection of Microorganisms is a primary or secondary fibroblastase40. cultures, harvest the virus fluid thus obtained as inoculants, inoculate additional primary or secondary fibroblast tissue cultures, incubate the cultures for 4-9 days, and isolate the viral fluid and add a protective colloid. After 45 minutes, lyophilize. 2. The method of claim 1, wherein the protective colloid is from 2% to 5% of a mixture of gelatin, sucrose, tryptone and collidone relative to the viral fluid.