CH618091A5 - Method for the attenuation of virulent feline rhinotracheitis virus and use of this virus for the production of a vaccine - Google Patents

Description

The invention relates to the objects characterized in the claims.

Infectious viral cat rhinotracheitis is a common, serious, and especially serious disorder in cats that is caused by the cat herpes virus. This virus is the causative agent of feline viral rhinotracheitis (FVR = "feline viral rhinotracheitis"). The literature suggests that about half of respiratory infections in cats are due to this disease. This virus infects the epithelial cells of the nose, throat, trachea and eyes, causing epitheliolysis and necrosis. The symptoms that appear on the eyes mainly affect the conjunctiva, but ulcerative keratitis can also develop. The virus is eliminated from the nose, eyes and mouth during the illness. Infections caused by the FVR virus are often serious and are significantly fatal, especially in young animals. If pregnant cats are infected by the virus, abortion or generalized infections occur in the newborn animals. The transmission of the FVR virus to cats susceptible to the virus is generally done by intranasal instillation, e.g. B. by droplets expelled when sneezing or by direct contact, generally from nose to nose. Resistance in animals that have recovered from a natural or experimental infection is short-lived (1 to 3 months).

The cat causing rhinotracheitis virus was first isolated from infected cats; see. R. A. Crandell and F. D. Maurer, Proc. Soc. Exptl. Biol. And Med., Vol. 97 (1958), p. 487. The term cat rhinotracheitis (feline viral rhinotracheitis) was used by R. A. Crandell and E. W. Despe-aux, Proc. Soc. Exptl. Biol. And Med., Vol. 101 (1959), p. 494,

suggested. Since then, a number of papers have appeared confirming the isolation of the FVR virus from cats. In this work this virus is identified as belonging to the group of herpes viruses and transmission, epidemiology and histological properties of this disease are also described; see. J.L. Bittie et al., Amer. J. Vet. Res., Vol. 21 (1960) p. 547; R.A. Crandell et al., J.A.V.M. A "Nd. 138 (1961) p. 191; J. Ditchfield and I. Gri-nyer, Virology, Vol. 26 (1965) p. 504; R.H. Johnson and R.G. Thomas, Vet. Ree., Vol. 79 (1966), p. 188; R. C. Povey, Vet. Ree., Vol. 82 (1969), p. 335; T. E. Walton and I. H. Gil-lespie, Cornell Vet., Vol. 60 (1970), p. 232; Colloquium Report, J.A.V.M.A., Vol. 157 (1970), p. 2043; R. A. Crandell, J.A.V.M.A., Vol. 158 (1971), p. 922; and SI Bistner et al., JAVMA, Vol. 159 (1971), p. 1223. Furthermore, in “Advances in Veterinary Science and Comparative Medicine”, published by CA Brandly and CE Cornelius, Academic Press, Inc., New York, 1973, Vol. 17, pp. 201 to 224, an overview article by RA Crandell with the chapter heading "Feline Viral Rhinotracheitis (FVR)".

A number of attempts to produce a vaccine against the FVR virus have been reported in the literature. However, none of these attempts have been successful. Povey and Johnson, J. Small Anim. Pract., Vol. 11 (1970), p. 490, report that immunization attempts by intramuscular administration of a non-attenuated FVR virus vaccine failed. Although these authors observed that trials with trials using FVR virus vaccines that were inactivated by treatment with formalin and / 3-propiolactone were successful, they found that these vaccines did not significantly reduce disease incidence Cat colonies can be reached. So far, there are no effective vaccines to protect cats against the FVR virus.

The object of the invention was therefore to provide a vaccine against cat rhinotracheitis.

It was found according to the invention that the FVR virus on cat tissue cultures, preferably kidney and tongue tissue cultures, can be attenuated and that the virulence of the virus can be altered and reduced in such a way that disease symptoms no longer occur after parenteral inoculation.

Accordingly, according to the invention, a modified or attenuated strain of live FVR viruses is made available which, when administered parenterally, preferably intramuscularly to cats, brings about an immunization against the virulent cat rhinotracheitis, specifically according to the method defined in claim 1.

Furthermore, according to the invention, a vaccine is made available which is attenuated to such an extent that, when used, an antibody reaction is produced which is sufficient for long-term immunization of cats.

The vaccine produced according to the invention permits safe use and does not cause any disease in cats after parenteral administration. In addition, the modified FVR virus is not transmitted from vaccinated animals to other animals which come into contact with them. This eliminates the possibility of increased virulence of the virus after passage through an animal. This is a major advantage in combating the cat's Rhi notracheitis.

Living, virulent FVR viruses can be isolated by conventional methods from cats infected with viral cat rhinotracheitis and then identified in the usual way; [see. R. A. Crandell and F. D. Maurer, Proc. Soc. Exptl. Biol. And Med., Vol. 97 (1958), p. 487; J.L. Bittie et al., Amer. J. Vet. Res., Vol. 21 (1960), p. 547 and J. Ditchfield and I. Gri-

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nyer, Virology, Vol. 26 (1965), p. 504]. In general, the virus is obtained by dabbing the nasal and conjunctival membranes of infected cats with a damp, sterile, cotton swab. The virus is then placed in a suitable breeding medium with cat tissue. For replication and isolation, the virus is subjected to standard series passages. A preferably used medium is obtained from cortical kidney tissue from 8 to 12 week old kittens, which according to the method described by J. Youngner (Proc. Soc. Exptl. Biol. And Med., Vol. 85 (1954), p. 202) for monkey kidney cells described method has been trypsinized.

With regard to the preparation of the vaccines according to the invention, it was found that the attenuation and modification of the virulent FVR virus can be easily achieved by carrying out relatively few, at least 7, serial passages in cat tissue at incubation temperatures of 30 ± 2 °, preferably about 32 ° C. The virus preparation can be cleaned using standard limiting dilutions, for example using the tube or plaque method, during or after the series passages.

The FVR virus can be attenuated, for example, in the following cat tissue breeding systems: lung, testicle, kidney, thymus, tongue and embryonic fetal tissue, as well as cell lines, e.g. B. the cat kidney cell line according to Crandell (CrFK), cat tongue cells at the level of the third passage (Fc3Tg) and the cat neurofibrosarcoma cell line (FNFS). Cat tongue cell lines are particularly preferred.

The duration of the passages is chosen so that sufficient replication of the virus between the passages is possible. Periods of 2 to 7 days are preferred. The optimal duration of the passages can easily be determined by customary methods, for example by cytopathic observations, in which e.g. B. allows the virus to grow to a stage during a certain passage, after which a strong cytopathic effect (CPE) can be observed with continued incubation.

The fact that valuable vaccines are obtained at the indicated low temperatures is surprising in view of the known fact that the virus or its pathogenicity does not change during series passages in cat tissue at normal incubation temperatures of about 35 to 37 ° C. after 100 passages; see. R. A. Crandell, J.A.V.M.A., Vol. 158 (1971), p. 922. As can be seen, for example, from Example 2 below, the modification of the virus according to the invention can be achieved by serial passages at lower incubation temperatures.

The above-mentioned new method for attenuating virulent cat rhinotracheitis viruses (FVR viruses) from which injection vaccines for immunizing cats against viral cat rhinotracheitis can be carried out, for example, by inoculating with the virulent FVR virus. Introduce the virus into a liquid cat tissue nutrient medium that is not toxic to the virus, attenuate the virus by incubating at 30 ± 2 ° C. for 2 to 7 days and then separate an inoculum of the virus and subject it to further series passages in other cat tissue cultures, whereby a total of at least 7 passages can be made.

The virus preparations produced according to the invention can be diluted to adjust their effectiveness. In addition, stabilizers such as dextrose and lactose or other non-toxic substances can be added. The virus preparations can also be stored in a dried state, for example freeze-dried, and then formulated into liquid vaccines. Stabilizers that are used in the freeze drying of viruses have been developed by

W. A. Rightsel et al., Cryobiology, (1967), 3, p. 423 and D. Greiff et al., Advances in Freeze Drying, ed. L. Rey, Hermann, Paris, 1966, pp. 103 to 122. The vaccines can also be used in a mixture with other immunogenic cat vaccines. The examples illustrate the invention.

example 1

A sample of live, virulent FVR viruses, prepared according to the method described by J.L. Bittie, Amer. J. Vet. Res., Vol. 21 (1960), p. 547, has been grown and isolated, is placed on a single-layer cell culture of a diploid cat tongue cell line in tissue culture tubes measuring 16 x 125 mm. The cell culture tubes are prepared as follows:

The Fe3Tg line described by K.M. Lee et al., Cornell Veterinarian, Vol. 59 (1969), p. 539 is used as the tongue cell line. Each tube contains 1 to 2 ml of growth medium consisting of Eagle minimal medium (MEM) containing 10% fetal calf serum, 0.1% lac talbumin hydrolyzate, 30 units penicillin, 30 / <g streptomy-cin and 2.5 /.ig amphotericin is offset. 1 ml of the cat tongue cells (200,000 cells / ml) is added to each tube. If necessary, the pH is adjusted to 7.2 to 7.8 with sodium bicarbonate. The cells are allowed to grow in the tubes at about 35 ± 2 ° C until a single-layer cell culture is reached. The liquid is then poured off. Then 1 to 2 ml of a maintenance medium is added which has the same composition as the aforementioned medium, with the exception that 1 to 2% fetal calf serum is used. About 4 to 6 of these tubes are used per virus passage.

Each tissue culture tube is treated with the FVR virus inoculum. The thus inoculated tubes are left at a temperature of 30 ± 2 ° C until a cytopathic effect (CPE) can be observed under the microscope (after about 2 to 7 days). When the CPE reaches approximately 75 to 90% of the single-layer cell culture, the contents of the tube are harvested and samples of 0.2 ml each of the inoculum are subjected to a further 6 identical serial passages (a total of 7 passages). After the 7th passage, a standard limit dilution cleaning is carried out using Eagle minimal medium (MEM) supplemented with the aforementioned antibiotics (no serum) as a diluent, the incubation temperature being kept at 30 ± 2 ° C. After 7 days, the last tube that reacts positively with 75 to 90% CPE is harvested, this whole procedure is repeated twice with a total of 10 passages. Finally, an 11th passage with the purpose of increasing the volume is carried out by inoculating a 0.5 ml sample from the 10th passage onto flasks which contain single-layer cell cultures of diploid cat tongue cell cultures, by the above-described proliferation of tongue cells have been obtained. After the 11th passage, the total amount is harvested, identified and titrated according to standard procedures.

Depending on its titer, the vaccine obtained in this way can be diluted or stabilizers or other non-toxic substances can be added. If necessary, the vaccine can be dried or used in liquid form. All non-toxic liquid tissue culture media can be used to replicate and attenuate the virus. In addition to the aforementioned Eagles Minimal Medium (MEM) with additives, other non-toxic, liquid tissue culture nutrient media can also be used.

1 ml of the vaccine prepared as above, which titrates at 35 ± 2 ° C to a virus titer of 104'5 TCIDS0 / ml (50% value of the “tis-sue culture infective dose”; determined by the CPE)

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was administered intramuscularly to three sensitive cats. Two other cats serve as non-vaccinated control animals. All 5 cats had previously been found to be seronegative to the FVR virus. The disease (FVR) can generally be observed around the 4th to 9th day after normal contact or challenge with the virulent FVR virus. On the 4th day after vaccination, a pharynx test is carried out, which is negative in all 5 cats, which shows that no virus is emitted. The antibody titre of all 5 cats is less than 1: 2 before vaccination. One month later, immediately before the challenge, the antibody titre of the 3 vaccinated cats is on average 1:18 compared to less than 1: 2 for the 2 non-vaccinated control animals . One month after vaccination, all 5 cats are infected intranasally with virulent FVR virus, droplets of virus being added to each nostril (approximately 10,000 to 20,000 TCIDS0 / cat). The cats are observed for 2 weeks. The 3 vaccinated cats did not develop any disease or symptoms. In contrast, the 2 unvaccinated control animals develop severe FVR disease and show typical symptoms such as fever, watery eyes, runny nose, lack of appetite and general malaise. 2 months after the challenge, the antibody titer of all 5 animals is approximately the same (1:54), which indicates that the vaccinated animals are protected and the control animals have been successfully infected.

Example 2

Live, virulent FVR viruses are identified according to the method described by J.L. Bittie et al., Op. Cit., Described methods grown and isolated. The isolated product, which the researchers named “F-2”, is subjected to 37 successive series passages in primary cat kidney tissue cultures in roller tubes. The periods are 2 to 7 days each. The methods described by Bittie et al. described growth media used. The incubation temperatures are 35 to 37 ° C. After the 37th passage it turns out that the virus has retained a high proportion of its pathogenicity and is therefore unsuitable for immunization purposes. However, the virus can be attenuated to obtain a suitable vaccine by subsequent passages at lower incubation temperatures. Accordingly, after the 37th passage, 44 further series passages (a total of 81 passages) are carried out at an incubation temperature of approximately 32 ° C.

A 0.2 ml inoculum from the 81st passage with a titer of Iqs.o xCID50 / ml, calculated according to the Reed-Muench method, is then applied to single-layer cell cultures of diploid cat tongue cell lines in tissue culture tubes measuring 16 x 125 mm given. According to Example 1, 7 further series passages and then 3 limit dilutions and a passage to increase the volume are carried out, so that there is a total of 11 series passages. After the 11th passage, the entire raw vaccine is harvested, identified (by serum neutralization test with specific FVR goat antiserum) and titrated.

1 ml of the vaccine prepared according to Example 2 with a titer of 105.4 TCIDS0 / ml at 35 ° C. (determined by the CPE) is administered intramuscularly to three sensitive cats that are seronegative to the FVR virus. Two other cats serve as unvaccinated control animals. On the 4th day after vaccination, a throat swab gave a negative result in all 5 cats, which shows that no viruses are secreted. Two weeks after the original vaccination, one of the three vaccinated cats received a second 1-ml injection intramuscularly. One month after the first vaccination, all 5 cats are infected intranasally with virulent FVR virus. Thereby virus droplets are in each

Nostril administered (10,000 to 20,000 TCID50 / cat). During the next two weeks of observation, the three vaccinated cats showed no changes in contrast to the two non-vaccinated cats, who developed the symptoms of FVR disease. The results obtained show that vaccination with the vaccine causes a minimal antibody response, while two vaccinations produce a much stronger reaction. The following antibody titers are observed:

Table I

Antibody response

Before the 1 month after 2 months after vaccination the challenge vaccination

Control animal 1 0 0 1:54

Control animal 2 0 0 1:54

Cat A 0 1:18 1:54

Cat B 0 1:18 1:54

Cat C * 0 1:54 1: 162

* Cat C received a second 1 ml injection 2 weeks after the first injection.

Example 3

Larger amounts of vaccine material are produced after the 11th passage on cat tongue cells described in Example 2, the same method being carried out in the flasks described there with a total of 17 successive series passages on cat tongue cells.

500 ml of the virus material obtained after the 7th passage are added to 500 ml of N — Z-amine lactose glutamate stabilizer and then distributed in standard vaccine ampoules (titer 104'6 TCID 50 / ml). It is then freeze-dried in the usual way. For vaccination purposes, each ampoule is mixed with 1 ml of pyrogen-free, sterile, distilled water. To two sensitive cats each

1 ml of the vaccine thus prepared administered intramuscularly. Another unvaccinated cat serves as a control animal. A week later, the two vaccinated cats received an identical strengthening dose intramuscularly. 9 months after the first vaccination, all three cats are infected intranasally with virulent FVR virus. For this purpose, droplets of virus are introduced into each nostril (10,000 to 20,000 TCID 50 / cat). The cats are then observed for 13 days. The two vaccinated cats showed mild symptoms in 2 of the total of 13 days, while the non-vaccinated control animal showed severe symptoms in 6 of 13 days. One year after the first vaccination, all 3 cats are infected with the FVR virus as in the first time, with none of the cats showing the symptoms of the disease. It follows that the vaccinated cats have a long-term effect. The fact that the symptoms of the disease did not appear in the non-vaccinated control animal after the 2nd challenge was to be expected, since immunity occurred due to the natural infection caused by the 1st challenge.

Further proof of the long-term effects of the vaccines can be found in the following one-year challenge study. 1 ml of the vaccine prepared according to Example 3 is administered intramuscularly to 8 sensitive cats.

4 unvaccinated cats serve as control animals. In a previous test, all 12 cats were found to be seronegative against the FVR virus. A week later, the 8 vaccinated cats received an identical strengthening dose via the intramuscular route. One year after the first vaccination, 3 cats received a third identical dose by the intramuscular route. 5 days later, all 8 cats are

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tranasally infected with virulent FVR virus. Virus droplets are placed in each nostril (10,000 to 20,000 TCIDS0 / cat). The cats are then observed for 13 days. The results are summarized in Table II:

Table II

Clinical symptoms Strength of symptoms on day no

Control animal 1

9

strong

Control animal 2

8th

strong

Control animal 3

8th

strong

Control animal 4

9

strong

Cat A

3rd

light

Cat B

3rd

light

Cat C.

4th

light

Cat D

1

light

Cat E.

6

light

Cat F *

0

no

Cat G *

0

no

Cat H *

3rd

light

* Cats F, G and H received two vaccinations.

In addition to the above-described production of vaccines from live virulent FVR viruses which are obtained from infected cats, the invention also relates to the production of FVR vaccines using FVR viruses which have been modified by the above-described method. From an economic point of view, it is impractical to use the living, virulent, FVR virus, which is obtained from infected cats, as the starting material for the preparation of the vaccines, and then to subject it to the necessary number of serial passages, which ultimately results in the use suitable modified virus is obtained in vaccines. The invention therefore also relates to a method for producing FVR vaccines, in which a virus which has already been modified by serial passages is used as the starting virus. This means that a cultivation virus from already attenuated virus material is used.

Accordingly, the invention also relates to a method for producing a vaccine against viral feline rhinotracheitis, which is characterized in that an FVR virus which has been attenuated in the manner described above is combined by a sufficient number of serial passages at incubation temperatures of 32 to 37 ° C liquid cat tissue nutrient medium, which is not toxic to the virus, until the medium contains 104 to 107 TCID (infectious dose) of the attenuated virus per ml, and then harvested for liquid vaccines.

According to the methods described here, the modified FVR virus can easily be grown in large quantities and large concentrations. Using the modified FVR virus propagated on cat tissue cultures in concentrations of at least about 104, generally about IO4 to 107 and preferably about 104'5 to 106'0 TCID of the virus per 1.0 ml of the final vaccine results in parenteral administration 1 ml each of cats stimulates the formation of protective FVR antibodies, comparable to the antibodies that are formed in natural infections, but without the usual pathological symptoms of viral cat rhinotracheitis. The vaccinated cats are therefore resistant to an infection (challenge) with the disease-causing virus.

A significantly increased antibody response can be observed when a second and even a third or further boosting dose of the vaccine is administered parenterally. For example, it has been found that a second intramuscular injection, which is given one week after the first vaccination, produces favorable results. For best results, it is recommended that the second injection be given no earlier than about 2 to 3 weeks after the first injection. Excellent results can be obtained by taking a strengthening dose up to 12 months after the first vaccination.

It was also found that the enhancement of antibody formation can be achieved in addition to the aforementioned parenteral administration of vaccine boosting doses by intranasally infecting cats that were previously immunized by parenteral administration of the vaccines with the FVR virus, either an FVR virus modified by the method according to the invention or an unmodified virulent virus is used.

This intranasal instillation after parenteral vaccination generates a significantly higher antibody level that lasts for a long time. In addition, the protection of the animals treated in this way turns out to be stronger when infected with a virulent virus, as a result of which no clinical disease symptoms occur after infection with 250,000 TCIDS0 / cat of the virulent, unmodified FVR virus. In order to achieve optimal results, it is recommended to allow sufficient time to sensitize the cat after the initial parenteral vaccination so that at least a minimum level of immunity is achieved, as shown by increased antibody formation, before the animal is subsequently exposed to intranasal contact with FVR. Virus undergoes. The internas instillation is preferably carried out 2 to 4 weeks after the original parenteral vaccination, although good results can also be achieved if the instillation takes place up to 12 months after the parenteral vaccination.

Intranasal instillation is easily accomplished by inhalation of the FVR virus, either by spraying conventional oral sols into the nasal passages or by placing droplets in the outer nostrils or in the nasal passages. A corresponding concentration of the FVR virus either modified according to the method described above, or in its living, virulent, non-attenuated form) for intranasal instillation following the initial vaccination by parenteral administration is approximately IO3 to 106 TCID / ml.

The initial vaccination by parenteral route with subsequent contact with FVR virus (modified or not) via the respiratory tract represents a new method of immunization against viral cat rhinotracheitis. This causes a humoral antibody reaction and a local immunity of the respiratory tract in the animal, so that better protection against this disease is achieved. Effective, long-term protection against viral cat rhinotracheitis is thus achieved.

1 ml each of the vaccines prepared according to Example 2 with a virus titer at 35 ° C. and 105'4 TCIDS0 / ml is administered intramuscularly to three sensitive cats, who had previously been found to be seronegative to the FVR virus. Two other cats are used as unvaccinated control animals. 11 days after the first vaccination, one of the three vaccinated cats is given a second 1 ml injection intramuscularly. Approximately 1 month after the first vaccination, all three vaccinated cats and the two non-vaccinated control animals are subjected to an intranasal instillation with an appropriately diluted virus preparation of the living, virulent FVR virus. The head of the cats is held backwards and virus drops with a total of 250,000 TCIDS0 are used

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a 26 gauge needle and syringe inserted into the nostrils. The results summarized in Table III show a significantly higher antibody response and the absence of clinical disease symptoms when the virus is administered intranasally following parenteral vaccination.

Table III antibody responses are summarized in Table IV. It can be seen that the combination of parenteral vaccination and intranasal administration has a long-term effect.

Table IV Antibody Response

Titer for intranasal

Titer after the intranasal

Clinical symptoms after

10th

Instillation

Instillation *

the intranasal

Titer at the

Titer after the

Clinical

Instillation

intranasal intranasal

Symptoms after

Instillation

Instillation **

the intranasal

Cat A

1:

: 15

1: 290

mild

Instillation

Cat B

1:

: 15

1: 417

mild

Cat C.

1:

: 7

1:96

mild

Cat A *

1:54

1: 162

no

15

Cat D

1

: 22

1: 200

mild

Cat B

1:18

1:54

no

Cat E **

1

: 22

1: 200

no

Cat C.

1: 8

1:54

no

Cat F **

1:

: 46

1: 290

no

Control animal 1

<1: 2

1:54

strong

Cat G **

1

: 32

1: 200

very mild

Control animal 2

<1: 2

1:54

strong

Control animal 1

<1

: 2

1:66

strong

20th

Control animal 2

<1

: 2

1:22

strong

* Cat A received two injections.

** The measurements were carried out 1 month after the intranasal instillation.

1 ml injections of the vaccines prepared according to Example 3 (titer 104.6 TCIDSo / ml are administered intramuscularly to 7 sensitive cats, of whom it was previously determined that they are seronegative to the FVR virus. Received 15 days after all 7 cats received a second similar injection. Three of the vaccinated cats received an identical third injection 5 days before intranasal instillation with live virus. Two additional cats are used as unvaccinated control animals non-vaccinated control animals an intranasal instillation of an appropriately diluted viral preparation with live, virulent FVR virus, each cat being administered 250,000 TCID in the nostrils, the results

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* The measurements were taken 6 weeks after the intranasal instillation

** These cats received a total of 3 intramuscular injections.

1 ml injections of the vaccines prepared according to Example 3 (titer 104.6 TCID50 / ml) are administered to two sensitive cats, who have previously been found to be seronegative to the FVR virus. 15 days later, both cats are given a second similar injection. Approximately 9 months after the initial vaccination, both cats are given an intranasal instillation with an appropriately diluted virus preparation with live, virulent FVR virus. Each cat receives virus droplets in the nostrils with a total of 100,000 TCID per cat. 3 months later, all cats are infected intranasally with 250,000 TCIDso / cat live FVR virus. The results are summarized in Table V:

Table V

at

antibody

Antibody clinical

antibody

titer the titer for

Symptoms titer

intranasal

Time of

14 days

6 weeks

Instillation

Challenge after the after the

Challenge

Challenge

Cat A

1: 7

1:45

no

1: 138

Cat B

1: 7

1:66

no

1:96

The last three trials show that long-term immunization against FVR in cats can be achieved by first administering a vaccine parenterally with at least about 104 TCID of an attenuated FVR virus that is passed through at least 7 two to seven day series passages of cat tissue cultures in one liquid culture medium has been weakened at incubation temperatures of 30 ± 2 ° C, and then administered via the respiratory tract about 103 to 106 TCID of the FVR virus in living, virulent, non-attenuated form. Similar results are obtained when 55 of approximately 103 to 106 TCIDs of the FVR virus, which has been attenuated according to the invention, are administered via the respiratory tract.

Claims (6)

618 091 1. A method for attenuating virulent cat rhinotracheitis virus, characterized in that the virus is attenuated by at least 7 serial passages of cat tissue cultures in a nutrient liquid at incubation temperatures of 30 ± 2 ° C. 2. The method according to claim 1, characterized in that one introduces an inoculum of live, infectious cat rhinotracheitis virus into a liquid nutrient medium which is non-toxic to the virus and contains living cat tongue cells, the virus by incubation at temperatures of 30 ± 2 ° C increased for 2 to 7 days and then an inoculum of this virus is separated and subjected to further series passages on other cat tongue cultures, with a total of at least 7 passages being carried out. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized in that live, infectious cat rhinotracheitis virus is weakened up to a virus titer of at least IO4 TCID per ml and the liquid vaccine is harvested. 4. The method according to claim 3, characterized in that cat tongue cells are used as the tissue culture. 5. attenuated cat rhinotracheitis virus obtained by the method according to claim 1. 6. Use of the attenuated cat rhinotracheitis virus according to claim 5, for the production of a vaccine, characterized in that the virus is subjected to serial passages of cat tissue cultures in a nutrient liquid at incubation temperatures of 32 to 37 ° C until a content of 104 to 107 TCID of the attenuated virus per ml has been reached, and the liquid vaccine is harvested.