RU2215031C1 - Strain feline herpesvirus-1 used for control of immunogenic activity of vaccine, preparing specific curative-prophylactic and diagnostic biopreparation - Google Patents

Abstract

FIELD: veterinary virology. SUBSTANCE: strain Feline herpesvirus-1 is deposited in collection of microorganisms VGNKI of veterinary preparations in 1994 year and has registration number "Grand-31". The strain shows high biological, antigenic and immunogenic activity and useful for preparing on its base highly immunogenic vaccine against infectious rhinotracheitis in cats, preparing hyperimmune sera in animals of different species for treatment, prophylaxis and diagnosis of infectious rhinotracheitis in cats and for control of these biopreparations. EFFECT: enhanced and valuable properties of strain and vaccine. 15 ex

Description

 The invention relates to the field of veterinary virology, in particular to a new strain of Feline herpesvirus-1, 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 herpesvirus.

 According to literature data abroad, various authors isolated Feline herpesvirus-1 strains at different times, but they do not provide detailed characteristics and areas of use of feline herpes virus strains (1, 2). In Russia, there are no reports of the isolation of Feline herpesvirus-1 strains from cats or the purchase of imported virus strains.

 The aim of the invention is to obtain a culture strain of feline herpes virus with stable infectious, antigenic and immunogenic activity, which can be used to control the immunogenic activity of vaccines, the manufacture of specific therapeutic and diagnostic biologics.

 The Feline herpesvirus-1 strain was obtained from the Feline herpesvirus-1 virus isolate isolated in 1994 from a dead cat of 4 months of age with signs of acute respiratory disease 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 All-Russian State Research Institute for Standardization and Certification of Veterinary Medicines and has a registration number - "Grand" -31.

The strain Feline herpesvirus-1 VGNKI "Grand" -31 is characterized by the following features and properties:
Morphological properties
Virions have cubic symmetry, the capsid contains 162 hollow capsomeres enclosed in a lipoprotein membrane. The diameter of the virion ranges from 120 to 173 nm.

Cultural properties
The virus is reproduced with a cytopathic effect in many primary and transplantable cell cultures, but the most sensitive to the virus are cultures: CrFk (cat kidney), Fs (cat spleen) and other feline cells. In these cell cultures, a pronounced cytopathic effect (CPP) occurs within 48-72 hours after infection. In feline cell cultures, the virus titer reaches 6.7 lg TCD 50 / cm ³ . The first signs of the cytopathic effect of the Grand strain virus in the cultures used is already visible 24 hours after infection and is manifested by the formation of individual foci of large round cells with the subsequent degeneration of the entire cell monolayer and its separation from the glass.

Antigenic properties
The strain "Grand" of herpesvirus cats with parenteral administration to rabbits induces the formation of neutralizing antibodies in titers of 5-8 log ₂ .

Virulence
With oronasal and intratracheal infection of intact cats with a culture seed of the Grand strain with an activity of 10 ⁷ TCD 50 / cm ³ at a dose of 1 cm ^3, clinical signs of herpes virus infection appear in all animals on days 4-7. In animals, there is a decrease in appetite, cough, rhinitis, catarrhal conjunctivitis, ulcerative stomatitis, fever (body temperature rises to 40.0 ^o C. On 8-9 days from the onset of clinical signs of the disease in 70% of cases there is a death, in 30% - clinical recovery of animals, with about half of the animals remaining virus carriers.In pregnant cats, the disease may be accompanied by abortion.

The stability of the basic properties of the strain
The strain of herpesvirus cats "Grand" retains the original infectious and antigenic activity for 10 passages.

Serological properties
Active in neutralization reaction with specific serum.

Hemagglutinating properties
The strain of herpesvirus cats "Grand" weakly agglutinates red blood cells of cats.

Safety for laboratory animals
The strain of herpesvirus cats "Grand" is harmless to white mice, guinea pigs and rabbits with intramuscular, subcutaneous and oronasal administration.

Additional strain information
The strain of herpesvirus cats "Grand" is free from bacterial, mycoplasma, fungal and viral contaminants.

The ability of the strain to spread in vivo
Infection with herpesvirus animals in the environment occurs aerogenically and through the contact of healthy animals with the secretions of patients. The incubation period is from 2 to 10 days.

Basic storage conditions
The strain of herpesvirus cats "Grand" 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 strain of herpesvirus cats "Grand" is used to study its virulent properties for cats. A virus with an activity of 6 lg TCD 50 / cm ³ is administered to 2 groups of kittens of 2-6 months of age oronasally 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 herpes virus antigen with an activity of 100 TCD 50 / cm ³ . Intact kittens become ill 3-5 days after infection with the clinical picture characteristic of viral respiratory diseases: fever, rhinitis, conjunctivitis, pneumonia. Kittens, in the blood serum of which the titer of humoral antibodies is 3 log ₂ and above, do not get sick under these infection conditions.

Example 2
Research is being conducted on the safety of the Grand herpesvirus strain of cats for white mice, guinea pigs and rabbits. A virus with an activity of 6 lg TCD 50 / cm ³ is administered oronasally to 3 groups (4 seronegative animals in each group): white mice, guinea pigs and rabbits in volumes: 0.3, 0.5 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 being carried out to study the stability of the virulent properties of the Grand herpesvirus strain of cats after 12 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 less than 6 lg TCD 50 / cm ³ . Herpesvirus-negative kittens of 3-5 months of age are injected with oronasal virus, 1 cm ³ once. Kittens become ill 4-7 days after infection with the clinical picture characteristic of viral respiratory diseases: fever, rhinitis, conjunctivitis, pneumonia.

Example 4
Studies are being conducted to study the stability of the Grand herpesvirus strain of cats when stored in lyophilized form. Herpes virus with an activity of 6.0 lg TCD 50 / cm ^{3 is} mixed with the drying medium VGNKI-3 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 herpesvirus during drying on the VGNKI medium is 0.5 lg TCD 50 / cm ³ . The decrease in the titer of herpes virus during storage for six months at a temperature of minus 20 ^o C is 0.1 lg TCD 50 / cm ³ , during the year - 0.2 lg TCD 50 / cm ³ , one and a half years - 0.5 ± 0.1 lg TCD 50 / cm ³ , two years - 1.0 ± 0.25 lg TCD 50 / cm ³ .

Example 5
The antigenic properties of the inactivated Grand herpesvirus strain of cats 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 6.5 lg TCD 50 / cm ³ inactivate formalin, the final concentration of which is 0.2%. To five cats of 4 months of age, an inactivated antigen in a volume of 1 cm ³ is administered subcutaneously once. The maximum accumulation of antibodies in serum of cats is recorded on 21 days after its administration. During this period, they gradually increase. The titer after a single injection of antigen in RNGA is 5-6 log ₂ .

Example 6
Specific hyperimmune sera for the Grand herpesvirus strain of cats 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 with 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 feline panleukopenia virus. The activity of the blood serum of rabbits obtained by weekly immunization is 5.0-6.0 log ₂ (PH), goats - 4.0-6.0 1og ₂ . Blood serum, which is obtained from rabbits and goats immunized with small doses of antigen, is 6.0-7.0 1og ₂ and 7.0-8.0 1og _2, respectively, with higher pH activity. The obtained sera do not show activity in pH with the panleukopenia virus of cats. In rabbits and goats in the control group, they are negative.

The obtained hyperimmune sera are used for serodiagnosis of infectious rhinotracheitis and for 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 specific hyperimmune goat blood serum with a titer of 6.0 log ₂ in pH, as well as cat serum with a titer of 8.0 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, and the serum of cats is 9.25 log ₂ .

The obtained globulins are used for the diagnosis of infectious rhinotracheitis 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
The feline herpesvirus diagnostic serum obtained as described in Example 6 is used to identify virus isolates isolated from cats with signs of respiratory illness. For this, the contents of the ampoule with lyophilized serum are dissolved in 10 cm ^{3 of the} growth culture medium, heated for half an hour at 56 ^o C in a water bath. The investigated samples of herpesvirus isolates and a control sample of herpesvirus cats strain "Grand" (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 CPD of isolates containing herpesvirus cats, which is manifested by the absence of CPD in tubes containing a mixture of virus and positive serum. In test tubes containing the same serum-free herpesvirus isolate, 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 herpes virus, a characteristic CPD does not appear in test tubes with this isolate, and in the case of the appearance of non-specific CPD, it is also observed in test tubes containing this isolate and anti-herpesvirus serum. In control tubes with an uninfected cell culture, no degenerative changes in the cell monolayer are observed.

Example 9
The antigenic activity of herpesvirus is evaluated in the Triket vaccine 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 virus-neutralizing antibodies to feline herpesvirus, the obtained sera are examined in pH with 100 TCD 50 / cm ^{3 of} the Herpesvirus strain Grand in a transplanted Fs cell culture. As a positive control serum in PH, a specific diagnostic serum for feline herpes virus was used, obtained as described in Example 6. The reaction was 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: 16-1: 128. When comparing the results of titration of sera obtained from cats before and after vaccination, it is noted that in all experimental animals the titers of specific antibodies to the causative agent of infectious rhinotracheitis 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 herpes virus component in the Triket vaccine is active and, therefore, the vaccine for this component is suitable for use.

Example 10
The vaccine against infectious rhinotracheitis of cats is obtained by culturing herpesvirus in a monolayer of kitten's kidney cells.

For infection, a culture herpesvirus is used. "Grand" 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 6 lg TCD 50 / cm ³ . The virus is inactivated by formalin, the final concentration of which is 0.2%. As an adjuvant, add a 3% solution of hydroxal in an amount of 10% by volume.

Inactivated feline rhinotracheitis vaccine is a pink or lilac-pink liquid with a loose sediment 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.

 The prepared vaccine is sterile in mycotic and bacterial respects.

Example 11
Obtained in example 10, a vaccine against rhinotracheitis of cats is checked 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.

Example 12
The vaccine against infectious rhinotracheitis of cats obtained in Example 10 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) remain normal, no local reaction to the introduction of the vaccine is noted. The drug is harmless.

Example 13
The immunogenic properties of the vaccine against infectious rhinotracheitis of cats obtained in Example 10 are tested. 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 herpes virus - the Grand strain with an 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 the clinical picture characteristic of viral respiratory diseases: fever, rhinitis, conjunctivitis, pneumonia. The general clinical condition of 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 14
The efficacy of the vaccine obtained in Example 10 is studied under clinical conditions. 32 cats and 24 cats are immunized with this vaccine, of which 19 are not purebred, 13 Persian, 10 Scottish fold, 9 British, 3 Siamese, 2 Devon Rex. The age of the animals is from 2 months to 5 years, with 75% of vaccinated animals under the age of 1 year. The vaccine is administered subcutaneously at 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 inhibition, 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 infectious rhinotracheitis during the year of observation.

Example 15
The medicinal properties of hyperimmune sera obtained in Example 6 and globulins obtained in Example 7 are studied. 45 cats of various breeds and ages with a diagnosis of “infectious rhinotracheitis” confirmed in PH are used in the experiment. In 12 cases, only antibiotics are used for treatment, in 12 cases, along with the use of the same antibiotics, non-specific treatment is supplemented with the use of hyperimmune serum (obtained in example 6) with a titer of 8.0 log ₂ , in 10 cases - globulin (obtained in example 7) from serum of goats, in 11 - 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 after 24-72 hours, which is 3-5 days earlier than usual, 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, which is manifested 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 effectiveness of the use of specific sera and globulins in the treatment of infectious rhinotracheitis in cats.

 The use of the strain Feline herpesvirus-1, VGNKI "Grand" -31 in veterinary practice will make it possible to judge with sufficient accuracy the quality of the manufactured and used biological products against herpesvirus infection in cats, to produce a highly immunogenic vaccine against infectious rhinotracheitis of cats on its basis, and to receive hyperimmune sera on different types of animals for the treatment and prevention of infectious rhinotracheitis in cats. Use antigen and serum obtained on it in serological reactions for the diagnosis of infectious rhinotracheitis in cats.

Sources of information
1. Arentt BD, Greene CE Feline respiratory disease / The book: Clinical Microbiology and infectious diseases of the dog and cat (ed. Greene CE) Philadelphia. - 1984.- p. 527.

 2. Gillespie J. H., Scott F.W. Feline Calicivirus (FCV) infection. // Adv. vet. sci. and comp. med. - 1973. - 17. - p. 176-188.

Claims (1)

 The strain Feline herpesvirus-1 VGNKI "Grand" -31, used to control the immunogenic activity of vaccines, the manufacture of specific therapeutic and diagnostic and biological products.