DE2544510A1 - Feline rhinotracheitis vaccine - by attenuation of live virus by serial passage at 25 degree C in cat kidney cells - Google Patents

Abstract

The attenuated, antigenic cat herpes virus (a) virulent live herpes virus is introduced into a tissue culture, (b) the culture is incubated until evidence of cellular cytophatology is apparent, (c) the virus is collected and introduced into a new tissue culture, (d) serial passage of the virus, in tissue culture is conducted a sufficient number of times at an incubation temp. sufficient to produce a non-virulent live antigenic virus, (e) the attenuated virus is siolated. Used in the treatment and prevention of viral rhinotracheitis in cats which is specific for deoxyribonucleic acid of cats. Young cats may be inoculated with vaccine prepd. from an attenuated virus, either by intraocular or intra-auricular admin. while matl. antibody is still present.

Description

"Viral Rhinotracheitis Vaccine for the Cat Sex and Procedure for its production.

The invention relates to a virus that affects cats 'tFeline rhinotracheitis "(F.V.R.) causes a herpes virus in cats and to a method of attenuating to an avirulent antigenic active form. In particular, the invention relates to a method of attenuating herpes virus in the cat sex by serial passage of the same through a tissue cell culture during a number of times and under such incubation conditions that the Virus becomes avirulent, i.e. it loses its virulence but its antigenic Maintains character.

BACKGROUND OF THE INVENTION F.V.R. is a specific herpes virus infection in the sex of cats. Herpes virus, especially in cats and closes in the epithelial cells that cover the nose, pharynx, and trachea disguise, and cause necrosis, sneezing, coughing, and nasal discharge. The epithelium the conjunctiva is also attacked, causing serious conjuctivitis. The bronchi and the larger bronchioles can sometimes become infected as well.

Sometimes the epithelium of the tongue can also be attacked, leading to causing inflammation of the tongue. The disease is characterized by developing conjunctivitis, eye discharge, nasal discharge, and sneezing. Since its first isolation, the herpes virus has been found to be common present in the normal cat sex.

F.V.R. virus is a DNA virus that is specific for the genus. The Folidas family is the only innkeeper from whom this F.V.R. virus has been isolated is. The virus can be found in cat tissue cells, but not yet in other systems. like other animal tissue or tissue cell cultures of other species, incubated eggs or. Experimental animals to be bred. The virus creates one specific cytophathogenic effect in tissue cultures. In addition, intranuclear Inclusion bodies can be detected in infected cultures.

The early stages of F.V.R. are characterized by an easy increased body temperature, sneezing, salivation, and serous eye discharge. Depending on the disease process progresses, the body temperature increases; the infected The cat becomes depressed and profuse eyes and mucous membranes develop Nasal discharge. Scabs and necrosis develop around the eyes and nose (of the epithelium and bone of the nasal concha). Leukocytosis develops, the total leukocyte count varying from 30,000 to 50,000 per cm3. The most obvious pathological changes are limited to the upper airways. Usually occurs no pneumonia except when bacterial or mycoplasma happens by chance available.

Tests on gnotobiotic kittens have shown that the Pathogenicity of herpes virus in the feline sex not from microbial flora depends on the airways. Gnotobiotic cats that use through their nasal passages lferpesvirus, develop a fever and severe upper respiratory infection caused by nicening, coughing, nasal discharge and eye discharge, discomfort, Anorexia, depression, and weight loss are characterized. The epithelium of the concha nasalis experiences necrosis in addition to epithelial changes in the nasal concha osteolytic changes clearly develop. It turns out that herpes virus preferentially reproduces in cats and necrosis in the Brings about growth zones of the bones.

There is much clinical and experimental evidence after the herpes virus infections in humans and in low animal species in-.

sexual tract infection, and congenital infections are known in many animal species. Experimental evidence shows that herpes virus Can and can induce infection of the genitals in the family of cats that the virus can cause serious congenital disseminated infection in kittens may that are born of mother cats that are in the later stages of pregnancy have been infected. Abortion was through both intravenous and intranasal Vaccination of mother cats during the later stages of their pregnancy. Vaginal infection of pregnant mother cats, vaginitis and fatal systemic Infection in the neonatal kittens has also been induced experimentally. Infected boy Cats had hepatic inclusions and pneumonia.

Although g.V.R. has long been considered a serious disease of the feline family is known and much time and attention has been devoted to this disease, there is currently no known vaccine to control and / or prevent this infection to disposal.

The invention aims to produce a vaccine, the genera of Felidae family against F.V.R.

will immunize which vaccine by attenuating a virulent Strain of herpes virus to an avirulent state while maintaining its antigenicity will be produced.

SUMMARY OF THE INVENTION.

In short, the invention relates to a vaccine that by a method is produced comprising the following steps: Isolation of a pure virulent kerpesvirus from the feline sex, inoculation of a tissue cell culture with the virus so that reprsduction of the virus will occur, serial passage of the virus thus propagated by additional tissue cell cultures during a sufficient period Number of times and under such conditions that the virus becomes an avirulent Condition is attenuated without losing its antigenic character.

Attenuation or modification of the virulent or pathogenic character from bacteria or a virus to an avirulent or non-pathogenic state is known.

Modification or attenuation of borphology and / or pathogenicity was effected by many techniques. Sometimes repeated series pass attenuates the germ of the disease through the host tissue; Serial passage through a fabric that is different from the host tissue sometimes leads to attenuation; more chemical Shock of the organism, radiation treatment, passage at low temperature and other techniques have been used by bacteriologists and the virologon, to obtain pathogenically inert organisms that retain their ability to cause ' that their innkeepers produce antibodies that effectively neutralize a germ can. Attenuation without loss of antigenicity is a living organism Not at all predictable and only empirical methods can be used to determine the expediency a given technique can be used.

A specific technique that can be used, for example, with a type of virus has been shown will not always be useful for a different type of virus. Until now is not a known method of attenuating herpes virus at Cats described. On one particular attempt, the virus did not even get through 150 passages attenuated through cat tissue; the final virus that will turn into a noninmlune kittens introduced were found to have a level of virulence which was not less than that of the starting material.

According to the invention it has now been found that if a tissue cell culture, vaccinated with a herpes virus in cats and a greatly reduced temperature, e.g. within the range of 23 - 330C, the virus attenuates and becomes avirulent, but still retains its antigenic character, i.e. if it is exposed to a sensitive Cat is introduced to its immunological mechanism for generating viral Will stimulate antibodies. The cat then becomes immune to the following infections.

Using standard sorting procedures, it has been found that the desired avirulent character without loss of antigenicity after about Developed 50 cycles at a temperature in the range of about 230C to about 330C Has. However, up to about 250 or 300 passes or more can be used.

According to a second aspect of the invention, an avirulontic antigen Virus as a virulent Herpes virus in cats, the F.V.R.

brought about can be obtained by a technique that does the following Steps include: growing an isolated virus on a tissue cell culture, E.g. a cat renal govebez ellenculture that the growth of the virus up to the Maintain desired viral titer by vaccinating a single layer the cell culture with the virus using the known "plaque" technique, being the only layer under a semi-solid nutrient solution, usually in one Vessel of the petri dish type, incubating at 250C until maximum cellular degeneration is proven (usually after about 10 to 12 days) that selecting the small, swollen, irregularly shaped spots that are next to regularly shaped non-swollen spots are present, and passing them one to three times these small spots in a monolayer tissue cell culture in the usual way as well harvesting this virus after the occurrence of maximal cellular degeneration.

This selective technique makes a minimum number of passes receive an avirulent antigenic virus.

It could be assumed that the incubation is at a low temperature a mixture of more strongly attenuated and iii less mass attenuated Colonies or spots of the virus result in which, by selecting the small ones, you want to remove them looking and irregular spots or 'colonies' created by the plaque' technique allows a more attenuated virus to be obtained without a continuous one Passage at low temperature is necessary to keep everything virus in the original Attenuate inoculants.

The F.V.R. vaccine according to the invention can be of any genus of the family of the Felidae by extraparenteral or intraparenteral techniques. The vaccine in liquid form can be administered intranasally, intraaurally, intraocularly, intramuscularly, administered intravenously, intraperitoneally, and the like. In dry In powder form, it is suitable for extraparanteral administration. As with vaccines this type is common, about 3000 to 20,000 TCID50 units will be in a single one Dose administered.

A distinctive feature of F.V.R. virus that is believed to That it is unique is that even those kittens that have high titers of maternal antibodies, i.e. kittens from birth to one About five weeks old, in the epithelial tissues of the nasal and eye canals with Are infected with the herpes virus. It will assumed that this Condition can result from the fact that the virus has a tendency to sting in the epithelial tissue to localize where the circulating antibodies in the bloodstream do not carry the virus can neutralize, and from there slowly spread, the particles can be made independent by the maternal antibodies. If later the level decreases in maternal antibodies in the kitten, the virus can spread systemically infect the kitten, usually leading to F.V.R. leads.

With the vaccine according to the invention by maternal antibodies protected young cats against F.V.R.infection by extraparenteral techniques, such as intraaural or intraocular administration of the vaccine. After the disappearance of the maternal antibodies can either be extraparenteral or parenteral, such as intramuscular, intravenous, intraperitoneal, administration techniques of the virus vaccine should be applied. As intranasal, intraaural or intraocular Administration of the vaccine in both maternal antibody protected young as well as adult cats, this vaccination technique is used for vaccination of young animals that are believed to still contain maternal antibodies available. In this stage is the determination of the maternal Antibody titer not necessary.

Detailed description of the invention.

The steps of the process of the present invention can be more clearly understood as follows be circumscribed: 1. Isolation of the virus From a cat whose upper respiratory tract infected and exhibiting the characteristic symptoms of F.V.R. exhibited, became made a line according to the standard technique. Material of the stroke was made into a Cat kidney tissue cell culture introduced and feline herpesvirus was purified according to the standardized immunological techniques using a 'monospecific antiserum for the virus isolated and identified.

2. Reproduction of the virus Tissue cell cultures were done in cats kidney cells produced. A standardized glass culture vessel was trypsinized with a The suspension of cat kidney cells containing sufficient amounts of media to cover the cells to a depth of about 2 inches. The Eagles minimum Es-2 Sensitive Medium (MEM), Science 130: 432 (1959) with 5% fetal bovine serum used.

Any standard growth medium such as Lactalbumen, Ilydrolystate Medium in Hankt's Balance Salt However, Solution, Medium 199, etc., can apply Find. D-.e culture vessels were incubated at 370C for 1 to 3 days, after which a single layer of feline kidney cells was formed.

The vial was then isolated using the herpes virus standardized aseptic techniques and incubated after an incubation of 24 to 72 Hours at 17 ° C, the cytopathology of the cells was practically complete, indicating that the virus had reproduced itself titration according to the dilution technique reported a titer of about 10 TCID 50 / ml.

100 TCID50 / ml of this material became more extraparentally sensitive Administered to the cat. After 5 to 7 days, the clinical symptoms of a typical appeared Case of F.V.R. infection, indicating that the material was virulent I-lerpesvirus contained.

The table below gives a description of a typical F.V.R. infection.

Table of Typical F.V.R. Infection in Very Young Cats Date Observation 4/26/73 Cat N-5 gave birth to five apparently healthy kittens. Mother cat showed no special features. Her throat was streaked and contained how was found herpes virus, 2.5.73 Mother and five kittens were photographed and the kittens' throats streaked. the The kittens' eyes weren't open yet, and the herpes virus wasn't out young cats isolated.

5/18/73 The first clinical symptoms appeared in three young cats who had some discharge from their eyes. The kittens were photographed and their throats smeared which were found to be positive for herpes virus was.

May 23, 73 All five kittens had slimy eyes.

5/29/73 All five kittens had mucous eyes and Nostrils.

1.6.73 All five young cats had slimy eyes and Nostrils.

4,6.73 eyelids of all five cats by slimy-purulent Exudate tightly closed.

5.6.73 Seriously clogged eyes and nostrils of all five cats, difficult breathing through the mouth.

8.6.73 Very serious clinical picture of infection in all five young cats. The kittens were photographed and their coals were smeared. Positive for herpes virus.

6/10/73 A young cat dead - section picture post mortem - death as a result by F.V.R.

12.G.73 Two kittens dead - autopsy post mortem - death as a result by F.V.R.

6/13/73 A young cat dead - section picture post nortem - death as a result by F.V.R.

6/21/73 A young cat dead - section picture post mortem - death as a result by F.V.R.

3. ATTENUATION Using standard tissue culture flasks and the MEM growth medium of the type described above became unicellular layers made from feline kidney cells. About 100 TCID50 / ml of the isolated virus were taken introduced into the bottles and the inoculated bottles were kept at a temperature incubated at 250C. After about 8 to 10 days there was a small area of cell degeneration visible and became a second series pass with a small amount of the liquid Medium carried out. Incubation at 25 ° C was continued and after each series run cellular cytopathology increased.

After about 15 to 20 series passes, the cytopathology became complete reached after 3 to 5 days of incubation at 250C.

The attenuation was observed closely and in each case after 10 series runs susceptible cats were vaccinated with approximately 10 6 TCID50 / ml of the virus.

All cats infected in this way developed the typical one clinical pathology by F.V.R. up to about 50 series runs. Above this The serial passage level, symptoms gradually became less serious, from which it emerged that the virus through the incubation conditions at low temperature gradually got weaker.

To demonstrate that serial passage of the virus at normal incubation temperatures did not affect the virulence of the virus or almost did not, samples of the F.V.R. virus were used from the 60th and 103rd

Serra passage taken and at 350C intranasally sensitive cats administered in an amount of 0.5 ml of the virus with a titer of 106 TCID50 / ml.

The cats were carefully examined and their temperatures recorded rectally. The data on this series are given in Tables I and III below. TABEL I (run 60 at 35 ° C) ate o. Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 09 temp. in ° F 102.4 103.0 103.8 104.4 104.0 103.8 103.0 102.4 102.6 102.8 102.8 103.0 102.4 Overall symptom 0 0 2 2 2 4 4 5 5 6 6 7 8 8 8 including 67 10 tem. in ° F 102.6 103.8 104.0 105.0 103.8 103.0 102.8 102.4 103.0 102.8 102.4 102.0 101.8 Symptom 0 0 0 2 2 2 3 4 5 5 6 6 6 7 8 56 11 Temp. In ° F 102.8 103.0 103.8 104.8 104.0 102.8 103.0 102.4 103.0 102.4 102.0 102.4 102.8 Symptom 0 0 0 1 2 2 2 3 4 5 6 6 6 8 8 53 12 Temp. in ° F 102.0 102.8 105.0 104.2 104.4 104.4 103.2 102.0 102.4 102.0 102.8 101.0 101.8 Symptom 0 0 0 1 1 2 2 2 2 4 4 5 6 7 8 44 The given count values have the following Meanings: 0 = none for F.V.R. - Symptoms characteristic of infection.

1 = less active, weak voice 2 = sneezing 3 = eye discharge - moist eyes; serous flow-fire nose 4 = decreased appetite 5 = practical no appetite - take some milk and water 6 = slimy-purulent eye exudate; slimy-purulent Neseexudart 7 = nasal congestion - breathing through the mind 8 = very sick - including symptoms 5, 6 and 7.

The cat does not react at all and looks pathetic and unkempt from 9 = cat dead TABLE II (Run 103 at 35 ° C) Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Ins-Temp. in ° F. 102.8 103.6 104.0 104.6 104.0 103.8 103.0 102.8 (NG) (NG) 102.0 102.8 101.8 ge Symptom 0 0 0 1 2 3 5 5 5 6 6 6 7 8 8 62 Temp. In ° F 102.0 104.2 104.6 103.8 105.0 104.2 102.8 101.6 (NG) (NG) 101.8 101.0 101.2 Symptom 0 0 0 1 1 2 3 3 5 5 6 6 7 8 8 55 Temp. In ° F 102.6 103.8 105.0 104.2 103.8 104.0 103.2 102.6 (NG) (NG) 102.4 102.0 103.4 Symptom 0 0 2 2 2 3 3 5 5 6 6 6 7 7 8 62 Temp. In ° F 103.8 104.4 104.0 103.6 104.8 104.0 102.8 103.6 (NG) (NG) 103.0 102.8 102.2 Symptom 0 0 0 1 2 2 3 3 3 5 5 6 6 7 8 51 NG = not done A Comparison of the data in Tables I and II shows that the virus was infected at pass 103 was practically as virulent as in run 60. So it is none or all too no attenuation of the virus received.

The effect. of the passage of the virus according to the invention indicated in the tables below.

The data show the results of intranasal administration of 0.5 ml 106 TCID50 virus after 48, 75 or 85 series runs, with the incubation temperature was kept at 250C. Examination of the data in the tables shows that the attenuation increases at the level of 85 series passes, TABLE III (Run 48 at 25 ° C) Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Total 7 Temp. In ° F 103.0 103.6 102.2 101.8 102.6 105.0 102.8 102.6 102.0 102.2 102.0 101.8 including Symptom 0 0 0 0 1 2 2 3 3 3 3 2 2 0 0 21 9 Temp. In ° F 103.2 104.2 104.4 104.0 103.2 103.0 102.6 102.8 101.8 102.0 102.4 102.2 Symptom 0 0 0 2 2 2 4 4 3 3 3 3 2 0 0 28 8 Temp. In ° F 102.8 103.4 103.8 102.8 103.0 013.2 102.8 102.4 103.0 102.4 102.2 102.4 Symptom 0 0 0 2 2 2 4 4 3 3 3 2 2 0 0 27 8 Temp. In ° F 103.0 102.8 103.4 103.0 102.8 102.8 103.0 102.6 102.4 102.8 102.4 102.2 Sumptom, 0 0 0 0 2 2 2 4 4 3 3 2 2 0 0 24 TABLE IV (Run 75 @ 25 ° C) t-day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Total 7 temp. In ° F 102.2 102.8 103.0 103.8 102.8 102.4 102.2 102.0 102.2 102.6 102.0 101.5 101.0 including symptoms 0 0 0 0 2 2 2 3 3 2 2 2 0 0 0 18 08 Temp. In ° F 102.2 102.6 103.6 103.0 102.8 102.4 102.4 102.0 102.8 102.4 102.2 102.0 102.2 Symptom 0 0 0 0 2 2 2 2 3 3 2 2 0 0 0 18 22 Temp. In ° F 102.0 102.4 102.8 104.0 103.2 102.8 102.4 102.0 102.0 102.4 102.2 101.8 102.0 Symptom 0 0 0 0 1 2 2 2 3 3 2 2 2 0 0 19 24 Temp. In ° F 102.8 102.6 103.6 103.0 102.6 102.4 102.4 102.6 102.2 102.0 101.8 102.0 101.8 Symptom 0 0 0 0 1 1 2 2 2 3 3 2 2 2 0 20 TABEL V (passage 85 at 25 ° C) t-day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 o.

Total 5K6 temp. In ° F 100.4 101.4 101.8 102.4 102.5 103.0 102.2 102.8 102.4 102.2 102.0 102.4 102.2 102.0 including symptoms 0 0 0 0 2 2 2 2 2 2 0 0 0 0 0 12 5K7 emp. in ° F 100.1 100.8 102.0 102.4 102.2 102.4 101.6 102.0 102.4 102.2 102.6 101.8 101.8 102.0 symptom 0 0 0 0 2 2 2 2 2 2 2 0 0 0 0 14 1K4 Temp. In ° F 101.6 102.4 102.0 102.8 103.0 103.6 104.6 103.0 102.8 102.4 102.0 102.2 102.0 102.4 Symptom 0 0 0 0 2 2 3 3 2 2 2 2 0 0 0 12 1K3 Temp. In ° F 101.0 102.0 102.4 102.6 102.6 101.6 102.6 102.4 102.0 102.4 102.2 101.8 102.0 101.8 Symptom 0 0 0 0 2 2 2 2 2 2 0 0 0 0 0 12 Cats infected with the virus in Tables III, IV and V vaccinated were then exposed to virulent virus and none of them developed F.V.R., suggesting that the avirulent virus had lost none of its antigenic effectiveness 4. I RSTELI, UNTG DES VAKZINS.

The attenuated virus prepared as described above was grown in large quantities using the growth medium described above used in standard flasks with a unicellular layer of feline kidney cells became. The flasks were incubated until the cytopathology was complete, which usually after 3 to 4 days. The growth medium was then harvested and mixed with a stabilizing solution in equal parts and in unit doses from about 1000 to 10,000 TCID50 / ml divided in the usual vaccine vial. That Material was then frozen to dryness in the vials and the vials were locked.

Although above the breeding and reproduction of the herpes virus Feline kidney cell tissue is described, it should be noted that: stable cell lines, derived from cat tissue can also be used. The stable ones Cell lines can be developed through repeated serial passage of cat tissue or can be obtained from commercial sources. Under the term "tissue cell culture" are here either primary feline kidney cells or from these cells understand derived stable cell lines.

In summary, the invention relates to a new vaccine for F.V.R. virus and a process for its production. The procedure includes the following steps: Introducing a live virulent herpes virus into a tissue cell culture; Incubate the culture until the cellular cytopathology is resolved clearly shows; harvesting part of the virus thus produced and reintroducing the harvested virus into a new tissue cell culture; repetition of the serial passage of the virus in Q; »fabric culture for a sufficient number Times at an incubation temperature that is sufficient to make an antigenically active, living one avirulent virus and recovering said virus from the called tissue culture medium. The preferred range of incubation temperatures used is between 23 and about 330C, with 250C being particularly preferred.

If the standard serial pass techniques are used, are about 50 passages are required to attenuate the virus to a sufficient extent. However, when the "plaque" techniques are used can be required attenuation can be achieved more quickly. Cat tissue, such as cat kidney cell tissue or a stable cell line derived from this tissue can be used for reproduction of the virus.

Claims (20)

Claims: 1. Process for the production of an antigen active live herpes virus in the feline sex, which includes the following steps: a) introducing a live virulent herpes virus into a tissue cell culture; b) incubating the culture until cellular cytopathologic is clearly evident; c) harvesting part of the virus ore in this way and reintroducing it the harvested virus into a new tissue culture; d) Repetition of series pass the virus in tissue culture for a sufficient number of times at an incubation temperature, sufficient to produce an antigenically active live avirulent virus, and e) recovering said virus from said tissue culture medium. 2. Method flat claim 1, characterized in that said Tissue cell culture a tissue cell culture of a member of the Felidae family is. 3. The method according to claim 1, characterized in that said Tissue cell culture is a cat tissue cell culture. 4. The method according to Änspruch 1, characterized in that that said tissue cell culture is a cat kidney tissue cell culture. 5. The method according to claim 1, characterized in that the incubation temperature for the series passes at a temperature in the range of about 23 to about 33 ° C is maintained. 6. The method according to claim 1, characterized in that the incubation temperature each row pass is about 25 ° C. 7. The method according to claim 1, characterized in that the series passage performed at least 50 times. 8. The method according to claim 1, characterized in that at least 50 series runs at an incubation temperature of about 23 ° C to about 3300 be performed. 9. The method according to claim 1, characterized in that said Tissue cell culture is a stable cell line derived from primary cat tissue is. 10. Method of producing an antigen active attenuated living Herpes virus comprising the steps of: a) introducing a live one virulent: herpes virus in a unicellular layer of a tissue cell culture that is derived from feline kidney cells; b) incubating the culture for about 3 to about 5 days at a temperature of 350C until cytopathology the cells are practically complete; c) harvesting part of the in this way produced virus and introducing the harvested virus into a freshly produced one unicellular layer derived from cat kidney tissue; d) incubation said culture at a temperature of about 25 ° C during a period the suffices to cytopathology at least a part of the cat tissue cells to lead; e) the passage in a row of a part of this. Way manufactured virus in cat tissue-derived cells for at least 50 times at an incubation temperature of about 25 ° C to obtain an antigenically active live avirulent virus, and f) recovering said virus from said last series pass. 11. Attenuated live herpesvirus virus in an effective amount of antigenically active avirulent lebandem herpesvirus produced by a process which comprises the following steps: a) introducing a live virulent IIerpesvirus in tissue cell culture; b) incubating the culture until clearly shows cellular cytopathology; c) harvesting a Part of the virus produced in this way and reintroduction of the harvested one Virus in a new tissue culture; d) repetition of the serial passage of the virus in tissue culture for a sufficient number of times at an incubation temperature, sufficient to produce a fairly active live avirulent virus; e) that Recovering said virus from said tissue culture medium, and f) adding said recovered virus to a stabilized solution, to make a vaccine out of it. 12. Vaccine according to claim 11, characterized in that said Tissue cell culture a tissue cell culture of a member of the Felidae family is. 13. Vaccine according to claim 11, characterized in that said Tissue cell culture is a cat tissue cell culture. 14. Vaccine according to claim 11, characterized in that said Tissue cell culture is a feline kidney tissue cell culture. 15. Vaccine according to claim 11, characterized in that the incubation temperature in the series passes at a temperature in the range of about 23 ° C to about 33 ° C is maintained. 16. Vaccine according to claim 11, characterized in that the incubation temperature each row pass is about 2500. 17. Vaccine according to claim 11, characterized in that the series passage performed at least 50 times. 18. Vaccine according to claim 11, characterized in that at least 50 series passes at an incubation temperature of about 230C to about 330C be performed. 19. Vaccine according to claim 11, characterized in that said Tissue cell culture is a stable cell line obtained from primary cat tissues is derived. 20. Attenuated live herpesvirus vaccine containing an effective amount of antigenically active avirulent live herpesvirus produced by the method comprising the steps of: a) introducing a live virulent herpesvirus into a unicellular layer of a tissue cell culture derived from feline kidney cells is; b) incubating the culture for about 3 to about 5 days at a temperature of 350G until the cytopathology of the cells is practically complete; c) harvesting a portion of the virus so produced and introducing the harvested virus into a freshly made unicellular layer derived from cat kidney tissue; d) incubating said culture at a temperature of about 2500 for a period sufficient to result in the cytopathology of at least a portion of the cat tissue cells; e) serial passage of a portion of the virus thus produced in cells derived from cat tissue for at least 50 times at an incubation temperature of about 25 ° C to obtain an antigenically active live avirulent virus, and f) recovering said virus from the opposite last rectification passage. 21. Methods of Protecting Sensitive Cats from herpes virus infection by putting said cats is administering an attenuated live virus vaccine that contains an effective amount of antigenically active avi- relentem live virus, which by the process ren, which comprises the following steps, a) the introduction of a live virulent herpes virus into a tissue cell culture; b) incubating the culture until the cellular Clearly shows cytopathology; c) harvesting part of the represented virus and the reintroduction of the harvested th virus into a new tissue culture;