RU2184567C1 - Strain feline calicivirus used for control of immunogenic activity of vaccines, preparing specific curative-prophylaxis and diagnostic biopreparations - Google Patents

Abstract

FIELD: veterinary science, veterinary virology. SUBSTANCE: strain Feline calicivirus shows the stable infectious, antigenic and immunogenic activities. Strain is used as a test-analysis of biopreparations against feline calicivirosis and ensures to prepare on its base the high-immune vaccine against feline calicivirosis, obtain hyperimmune sera for animals for treatment and prophylaxis of feline calicivirosis and use antigen and serum prepared from its in serological reactions for diagnosis of calicivirosis in cats. EFFECT: strain indicated above, valuable veterinary properties of antigen and vaccine.

Description

 The present invention relates to the field of veterinary virology, in particular to a new strain of feline calicivirus, used to control the immunogenic activity of vaccines, the manufacture of inactivated vaccines and the production of specific therapeutic and diagnostic sera and globulins and as an antigen for the detection of antibodies to feline calicivirus.

 According to literature data abroad, different authors at different times isolated strains of feline calicivirus. Calicivirus was first isolated and described by Fastier L. B. (1957) in New Zealand, Bolin (1957) in America, Burki (1963) in Sweden, Zwillenberg and Burki (1966) in Austria, Torlone (1960), Prydie (1966 ) in England, Studdert (1970) in Australia. Detailed characteristics and areas of use of none of these strains of calicivirus are given. In our country, we did not find reports of the isolation of feline calicivirus strains from cats or the purchase of imported virus strains.

 The aim of the invention is to obtain a culture strain of feline calicivirus with stable infectious, antigenic and immunogenic activity, which can be used to control the immunogenic activity of vaccines, the manufacture of inactivated vaccines and the production of specific treatment and prophylactic and diagnostic sera and globulins and as an antigen to detect antibodies to cat calicivirus.

 The feline calicivirus "Lars" strain was obtained from a isolate of the feline calicivirus virus from a sick mongrel cat of 3 months of age with signs of acute respiratory disease isolated in 1993 by successive passages on a subculture of cat kidney cells.

 In the same year, the strain was deposited in the collection of microorganisms of the State institution of the All-Russian State Research Institute for Standardization and Certification of Veterinary Medicines and has a registration number of 30 DEP.

The strain feline calicivirus strain "Lars" VGNKI 30 is characterized by the following features and properties:
Morphological properties.

 Virions have an icosahedral shape, shellless, reminiscent of the "Star of David". The diameter of the virions ranges from 37 to 40 nm.

 Cultural properties.

The virus multiplies well in cat kidney cell culture, transplanted cell cultures: CrFk (cat kidney), Fs (cat spleen) and other feline cells. Causes a cytopathic effect (CPP). When cultured in a primary trypsinized culture of cat kidney cells and in transfected cultures of CrFk, Fs cells, the virus titer is 6-8 lg TCD 50 / cm ³ . The cytopathic effect of the virus of the Lars strain in the cultures used is manifested 6-10 hours after infection by rounding a significant number of cells, their gradual degeneration and separation from glass with the formation of voids in the form of a "grid".

 Antigenic properties.

Strain "Lars" upon parenteral administration to rabbits induces the formation of virus-neutralizing antibodies in titers 6-9log ₂ .

 Virulence.

With oronasal and intratracheal infection of intact cats with culture seed strain at a dose of 10 ⁷ TCD 50 / cm ^3, clinical signs of calicivirus infection appear in all animals on days 3-6. In animals, there is a decrease in appetite, cough, rhinitis, catarrhal conjunctivitis, ulcerative stomatitis, fever (body temperature rises to 40.0-41.0 ^o C. On 8-9 days from the onset of clinical signs of the disease in 70% of cases there is a death, 30% of the clinical recovery of animals, with about half of the animals remaining virus carriers.

 The stability of the basic properties of the strain.

 Strain "Lars" maintains stable infectious and antigenic activity for 10 passages.

 Serological properties.

 Active in neutralization reaction with specific serum.

 Hemagglutinating properties.

 The Lars cat calicivirosis strain does not agglutinate animal red blood cells.

 Harmless to laboratory animals.

 The strain of calicivirosis cats "Lars" is harmless to white mice, guinea pigs and rabbits with intramuscular, subcutaneous and oronosal administration.

 Additional information about the strain.

 The strain "Lars" is free from bacterial, mycoplasma, fungal and viral contaminants.

 The ability of the strain to spread in vivo.

 Infection of animals in the environment occurs aerogenically and through the contact of healthy animals with the secretions of patients. The incubation period is 3-6 days.

 Basic storage conditions.

The strain "Lars" feline calicivirus lyophilized with stabilizing additives remains active at -20 ^o With at least one year.

 The invention is illustrated by the following examples.

Example 1
The Lars feline calicivirus strain is used to study its virulent properties for cats. A virus with an activity of 7 lg TCD 50 / cm ³ is administered to 2 groups of kittens of 2-6 months of age, oronasally and intratracheally at a dose of 1 cm ³ . Before infection, the blood serum of the animals is examined for the presence of specific antibodies in the pH in a kitten's kidney cell culture with a calicivirus antigen with an activity of 100 TCD 50 / cm ³ . Intact kittens become ill 3-5 days after infection in one way or another with the clinical picture characteristic of viral respiratory diseases: fever, rhinitis, conjunctivitis, ulcerative stomatitis, pneumonia. Kittens in the blood serum of which the titer of humoral antibodies is 5 log ₂ and above do not get sick under these infection conditions.

Example 2
Studies are being conducted to study the harmlessness of the Lars cat calicivirus strain for white mice, guinea pigs and rabbits. A virus with an activity of 7 lg TCD 50 / cm ³ is administered oronasally to 3 groups of 5 seronegative animals: white mice, guinea pigs and rabbits in volumes: 0.3 cm ³ , 0.5 cm ³ and 1.0 cm ^3, respectively. Observe infected animals for 14 days. During the observation period, all animals remain clinically healthy.

Example 3
Studies are carried out to study the stability of the virulent properties of the Lars cat calicivirus strain after 10 passages on cat kidney, CrFk (cat kidney), Fs (cat spleen) cell cultures. Spend 10 passages of the virus on these cell cultures and receive viral collections with activity not lower than 7 lg TCD 50 / cm ³ . For calicivirus seronegative kittens of 3-5 months of age, 1 cm ^{3 of the} virus is administered once oronasally. Kittens become ill on the 4th – 7th day after infection with the clinical picture characteristic of viral respiratory diseases: fever, rhinitis, conjunctivitis, ulcerative stomatitis, pneumonia.

Example 4
Studies are being carried out to study the stability of the Lars cat calicivirus strain when stored in lyophilized form. Calicivirus with an activity of 8.5 lg TCD 50 / cm ^{3 is} mixed with drying medium VGNKI in a ratio of 1: 1 and lyophilized. The activity of the virus is determined by titration in the culture of Fs cells before and after lyophilization and upon further storage at a temperature of minus 20 ^o With an interval of six months. The decrease in the titer of calicivirus during drying on the medium VGNKI is 0.2-0.5 lg TCD 50 / cm ³ . The reduction in titer of calicivirus during storage for six months at a temperature of minus 20 ^o C is 0.1 lg TCD 50 / cm ³ , during the year - 0.3 lg TCD 50 / cm ³ , one and a half years - 0.5 lg TCD 50, two years - 1.0 lg TCD 50 / cm ³ .

Example 5
The antigenic properties of the inactivated Lars cat calicivirus strain for cats are determined. To do this, a culture virus seed is obtained on the transplanted Fs cell line. Viral collection with a titer of 9 lg TCD 50 / cm ³ inactivate formalin, the final concentration of which is 0.2%. Five cats of 4 months of age are injected subcutaneously with a once inactivated antigen in a volume of 1 cm ³ . The maximum accumulation of antibodies in serum of cats is recorded on the 21st day after the administration of calicivirus. During this period, they gradually increase. The titer after a single injection of antigen in RNGA is 6-8 log ₂ .

Example 6
Specific hyperimmune sera for the Lars cat calicivirus strain are obtained in rabbits and goats. 2 methods of immunization are used: weekly 3-fold administration of antigen in a volume of 1 cm ^{3 to a} rabbit and 3 cm ³ goat and immunization with small doses of antigen, in which the antigen is administered intramuscularly in the same volume and the addition of Freund's complete adjuvant (1: 1), and then after 2 months without an adjuvant intravenously in a volume of 0.3 cm ³ and 0.5 cm ³ respectively. Blood sampling for obtaining serum samples is carried out 14, 21 and 28 days after the last injection of antigen. The activity of the obtained sera is set in pH. The specificity of the obtained sera is checked in pH with this virus and the PLC virus.

Using various methods of hyperimmunization of rabbits, all animals receive serum active in pH. In rabbits and goats in the control group, they are negative. The blood serum of rabbits injected with antigen on a weekly basis has activity in pH 6.0-7.5 log ₂ , goats 5.5-7.5 log ₂ . Blood serum, which is obtained from rabbits and goats immunized with small doses of antigen, is 7.6-8.5 log ₂ and 8.0-8.8 log _2, respectively, with higher pH activity. The obtained sera do not show activity in pH with the panleukopenia virus of cats.

The resulting hyperimmune sera are used for the diagnosis of calicivirosis in serological reactions and for the specific treatment of sick animals. Serum is stored in lyophilized or native form at a temperature of minus 10-20 ^o C for 24 months.

Example 7
Globulin is obtained from the blood serum of hyperimmunized goats with a titer of 8.0 log ₂ in pH and cats with a titer of 8.5 log ₂ in PH. To do this, use the generally accepted method of salting out with saturated sodium sulfate solution.

The globulin precipitate was resuspended in a phosphate buffer solution of pH 7.2. The titer of goat serum globulin after resuspension is 8.5 log ₂ in pH, for cats - 9.25 log ₂ .

The obtained globulins are used for the diagnosis of calicivirus in serological reactions and for the specific treatment of sick animals. Globulins are stored in a lyophilized or native state at a temperature of minus 10-18 ^o C for 24 months.

Example 8
Diagnostic serum for feline calicivirus, obtained as described in example 6, is used to identify isolates of viruses isolated from cats with signs of respiratory disease. For this, the contents of the ampoule with lyophilized serum are dissolved in 10 cm ^{3 of} growth culture medium. The test samples of isolates of calicivirus and a control sample of calicivirus cats strain Lars (positive antigen control) are combined with diluted serum in a ratio of 1: 1. The resulting mixture was incubated for 1 hour at room temperature and 1 cm ^{3 was} added to test tubes with a cat kidney cell culture per formed monolayer (4 tubes per sample). At the same time, 1 cm ³ dilution of isolates 1: 2 in a supporting culture medium without serum was added to 4 other tubes with cell culture. Control of cell culture are uninfected tubes with a monolayer of cells, where 1 cm ^{3 of} maintenance culture medium without serum and virus is added. All tubes are placed in a thermostat for 7 days and microscopic daily. Diagnostic serum neutralizes the CPP of isolates containing feline calicivirus, which is manifested by the absence of CPP in test tubes containing a mixture of virus and positive serum. In tubes containing the same serum-free calicivirus isolate, the CPD appears on the first day by a characteristic rounding of the cells, and then their rejection from the glass with the formation of voids in the form of a "grid". In the study of samples of isolates that do not contain calicivirus, a characteristic CPD does not appear in test tubes with this isolate, and in the case of the appearance of a nonspecific CPD, it is also observed in test tubes containing this isolate and anticalcivirus serum. In control tubes with an uninfected cell culture, no degenerative changes in the cell monolayer are observed.

Example 9
Assess the antigenic activity of calicivirus in the vaccine "Triquet" against calicivirus, infectious rhinotracheitis and panleukopenia of cats, manufactured by Nobivak, Holland, ser. 9515E. For this, cats 2-4 months of age are vaccinated with the indicated vaccine in a dose of 1 cm ³ according to the instructions for use. Before vaccination and after 21 days, all vaccinated puppies take blood samples and get blood serum.

To determine the titer of neutralizing antibodies to feline calicivirus, the obtained sera are examined in pH with 100 TCD 50 / cm ^{3 of} the Lars calicivirus strain in an Fs cell culture being transplanted. As a positive control serum in the pH, a specific diagnostic serum for feline calicivirus is used, obtained as described below in Example 6. The reaction is evaluated after 7 days for the development of CPD in cell culture. The titer of neutralizing antibodies in the blood serum of vaccinated cats is 1:32 - 1: 256. When comparing the results of titration of sera obtained from cats before vaccination and after, it is noted that in all experimental animals the titers of specific antibodies to the causative agent of calicivirus increase by at least 2 times, and in seronegative animals, before vaccination, they become no lower than 1: 32.

 Thus, it was found that the calicivirus component in the Triket vaccine is active and, therefore, the vaccine for this component is suitable for use.

Example 10
A feline calicivirosis vaccine is obtained by culturing calicivirus in a monolayer of kitten's kidney cells.

For infection use a culture calicivirus pc. "Lars" 2 levels of passages with activity in the pH 9 lg TCD 50 / cm ³ in the amount of 1 cm ³ to 200 cm ^{3 of the} supporting medium. The cultivation time is 48-72 hours. After this period, a single freezing and thawing of the virus-containing culture fluid is carried out. Before inactivation of viral collections, electron microscopy is performed to exclude their contamination and the infectious activity of the viral collection in the cell culture is determined. The activity of the virus in the pH should be at least 7 lg TCD 50 / cm ³ . The virus is inactivated by formalin, the final concentration of which is 0.2%. Hydroxal at a final concentration of 10% is used as adjuvant.

The inactivated feline calicivirosis vaccine is a pink or mauve pink liquid with a loose precipitate that breaks when shaken into a homogeneous suspension. It is suitable for use within 18 months from the date of its manufacture, subject to storage at a temperature of 2-4 ^o C.

 A vaccine is tested for bacterial and fungal contamination. To test the vaccine for sterility, crops are made on culture media MPA, MPB, MPPB, Saburo according to the generally accepted methodology. It is established that the prepared vaccine is sterile in mycotic and bacterial respects.

A vaccine is tested for antigenic activity. For this, 3-4-month-old seronegative kittens and rabbits weighing at least 3 kg of the vaccine are administered subcutaneously once at a dose of 1.0 cm ³ . Animal sera on the 21st day after immunization are examined in pH. The titer is 6-7 log ₂ . The vaccine is antigenic.

A vaccine is tested for safety. To do this, it is administered subcutaneously to 3 groups of white mice, respectively, 0.1; 0.25 and 0.5 cm ³ . Two groups of guinea pigs - 1.5 and 2.5 cm ^{3 each} . Two groups of rabbits - 2.5 and 5.0 cm ³ . Kittens - 1.0 (1 dose); 2.0 (2 doses) and 3.0 cm ³ (3 doses). The control animals were administered the maximum amount of culture fluid for this group without virus and adjuvant. Observe all animals for 3 weeks. The body temperature and general clinical condition of all experimental animals during the observation period (21 days) remains normal, no local reaction to the introduction of the vaccine is noted. The drug is harmless.

A vaccine is tested for immunogenicity in cats. For this, two groups of 4 seronegative kittens of 3-4 months of age are used. The first group of kittens is administered subcutaneously, once in a dose of 1.0 cm ³ . The second group of kittens (control) is introduced in the same volume of the culture medium without the virus. 21 days after the start of the experiment, all kittens are infected with oronasal calicivirus with activity of at least 7 lg TCD 50 / cm ³ , which is administered at a dose of 1 cm ³ . Intact kittens in the control group get sick 3-5 days after infection with a clinical picture characteristic of viral respiratory diseases: fever, rhinitis, conjunctivitis, ulcerative stomatitis, pneumonia. The general clinical condition of the vaccinated kittens during the observation period (21 days) remains normal, no local reaction to the introduction of the vaccine is noted. The vaccine is immunogenic.

Example 11
The efficacy of the vaccine obtained in Example 10 is studied under clinical conditions. 47 cats and 43 cats are immunized with this vaccine, of which 24 are purebred, 21 are Persian, 19 are Scottish fold, 14 are British, 5 are Siamese, 7 are Devon Rex. The age of the animals is from 2 months to 5 years, with 82 vaccinated animals under the age of 1 year. The vaccine is administered according to the instructions for its use subcutaneously in a dose of 1 cm ³ .

 In no case do not observe post-vaccination reactions of both local and general order. None of the animals noted oppression, refusal of food, allergic reactions. Local skin reactions to the introduction of the vaccine are not detected. In no case have post-vaccination complications and diseases of animals been observed with calicivirosis during the year of observation.

Example 12
The medicinal properties of the hyperimmune sera obtained in Example 6 and the globulins obtained in Example 7 are studied. 64 cats of various breeds and ages are used in the experiment, the diagnosis of "calicivirosis" in which is confirmed in pH. In 16 cases, only antibiotics are used for treatment, in 18 cases, along with the use of the same antibiotics, nonspecific treatment is supplemented with the use of hyperimmune serum (obtained in example 6) with a titer of 8.0 log ₂ , in 14 cases of globulin (obtained in example 7) from serum of goats, in 16 - globulin from serum of cats. Specific drugs are administered to cats subcutaneously, at 1.0 cm ³ per day, for 3 days. In all cases of using specific drugs, cats recover, which occurs within 24-72 hours, which is earlier than usual by 3-5 days, and in no case relapse or complications of the disease are noted. In the group of cats that are prescribed only antibiotics, at least 2 deaths are noted; in the group where serotherapy is carried out, fatal cases are not noted. In one Devon Rex kitten, an allergic reaction to the second administration of goat serum is recorded, expressed by flushing of the skin and itching. This reaction is eliminated by a single injection of a solution of diphenhydramine in generally accepted doses. With the introduction of globulins to kittens of the same litter, allergic reactions are not noted. Studies have established the therapeutic efficacy of the use of specific sera and globulins in the treatment of feline calicivirosis.

 The use of feline calicivirus "Lars" strain VGNKI 30 in veterinary practice will make it possible to fairly reliably judge the quality of manufactured and used biological products against feline calicivirosis, produce a highly immunogenic vaccine against feline calicivirosis on its basis, and obtain hyperimmune serums on different types of animals for the treatment and prevention of feline calicivirus , use the antigen and the serum obtained on it in serological reactions for the diagnosis of feline calicivirosis.

Claims (1)

 The strain Feline calicivirus VGNKI "Lars-30-DEP" used to control the immunogenic activity of vaccines, the manufacture of specific therapeutic and diagnostic biologics.