The strain of feline herpesvirus-1, used to control immunogenic vaccine manufacturer specific therapeutic and diagnostic biologicals
(57) Abstract:The invention relates to the field of veterinary Virology. The strain of Feline herpesvirus-1 deposited in the collection of microorganisms VGNKI veterinary drugs in 1994 and has a registration number "Grand"-31. The strain has a high biological, antigenic and immunogenic activity and suitable for manufacture on the basis of a highly immunogenic vaccine against infectious bovine rhinotracheitis cats receiving hyperimmune sera of different animal species, for the treatment, prevention and diagnosis of infectious bovine rhinotracheitis in cats and control of these biological products. The invention relates to the field of veterinary Virology, in particular to a new strain of Feline herpesvirus-1, used to control immunogenic vaccines, production of inactivated vaccines and achieve specific therapeutic and diagnostic serum and globulin and as an antigen to detect antibodies to the herpes virus cats.According to literature data abroad by different authors in different time allocated strains of Feline herpesvirus-1, but detailed characteristics and areas of use of strains of feline herpesvirus they are not what Tambov virus.The aim of the invention is to provide a culture of strain herpesvirus cats with stable infectious, antigenic and immunogenic activity, which can be used to control immunogenic vaccine manufacturer specific therapeutic and diagnostic Biologicals.The strain of Feline herpesvirus-1 obtained from a dedicated in 1994 isolate virus, Feline herpesvirus-1 from dead cats 4 months of age with symptoms of acute respiratory disease by serial passages on the subculture of the cells of the kidney of a cat.In the same year, the strain deposited in the collection of microorganisms of the all-Russian state research Institute for control, standardisation and certification of veterinary preparations and has a registration number is "Grand"-31.The strain of Feline herpesvirus-1 VGNKI "Grand"-31 characterized by the following features and properties:
The virions have cubic symmetry, the capsid contains 162 hollow capsomere, prisoners in lipoproteina shell. The diameter of the virion varies from 120 to 173 nm.Cultural properties
Virus reproducerea with the manifestation of cytopathic Auda culture: CrFk (kidney cats) Fs (spleen cats) and other cells of feline origin. In these cell cultures in 48-72 hours after infection occurs a pronounced cytopathic effect (JRC). In cell cultures of feline origin titer of the virus reaches 6,7 lg TCD 50/cm3. The first signs of cytopathic effect of the virus strain "Grand" in the cultures is evident as early as 24 hours after infection and is manifested by the formation of isolated pockets of large rounded cells with subsequent degeneration of the entire cell monolayer and separating it from the glass.Antigenic properties
Strain "Grand" herpesvirus cats in injecting rabbits induces the formation of neutralizing antibodies in the credits 5-8 log2.Virulence
When nasal and intratracheal infection of intact cats culture raspadkay strain Grand activity 107TCD 50/cm3at a dose of 1 cm3clinical signs of herpes infection are manifested in all animals for 4-7 days. The animals celebrate decreased appetite, cough, rhinitis, catarrhal conjunctivitis, ulcerative stomatitis, fever (body temperature is increased to 40.0oC. For 8-9 day since clkao half of the animals remain virus carriers. Pregnant cats, the disease may be accompanied by abortion.The stability of the basic properties of the strain
Strain herpesvirus cats Grand preserves the original infectious and antigenic activity for 10 passages.Serological properties
Active in the neutralization with specific serum.Hemagglutinins properties
Strain herpesvirus cats Grand poorly agglutinate erythrocytes of cats.Emissions laboratory animals
Strain herpesvirus cats Grand harmless for white mice, Guinea pigs and rabbits by intramuscular, subcutaneous and nasal administration.For more information about the strain
Strain herpesvirus cats "Grand" free from bacterial, mycoplasmal, fungal and viral contaminants.The ability of the strain to spread under natural conditions
Infection with a herpesvirus animals in the environment is airborne and through contact with healthy animals with secretions of patients. The incubation period from 2 to 10 days.Main storage conditions
Strain herpesvirus cats Grand dried with stabilizing additives Sokh the measures.Example 1
Strain herpesvirus cats "Grand" is used for studying the virulence properties for cats. Virus activity 6 lg TCD 50/cm3enter 2 groups of kittens 2-6 months of age oronasal dose of 1 cm3. Before infection of the blood serum of animals examined for the presence of specific antibodies in PH in the culture of kidney cells kitten with herpes virus antigen activity 100 TCD 50/cm3. Intact kittens sick 3-5 days after infection with typical viral respiratory disease clinical manifestations: fever, rhinitis, conjunctivitis, pneumonia. Kittens in the serum which titer humoral antibodies is 3 log2and above, do not develop under these conditions of infection.Example 2
Conduct research on the safety of strain herpesvirus cats "Grand" for white mice, Guinea pigs and rabbits. Virus activity 6 lg TCD 50/cm3enter oronasal 3 groups (4 seronegative animals in each group): white mice, Guinea pigs and rabbits in the volume: 0.3, 0.5 and 1.0 cm3respectively. Watch infected animals 14 days. During the observation period all animals remain clinically healthy.
3. Seronegative for herpes virus kittens 3-5 months of age, the virus is injected Gonzalo, on 1 cm3and one time only. Kittens sick 4-7 days after infection with typical viral respiratory infections clinical manifestations: fever, rhinitis, conjunctivitis, pneumonia.Example 4
Conduct research to study the stability of the strain herpesvirus cats "Grand" when stored in a dried form. Herpesvirus activity of 6.0 lg TCD 50/cm3mix with medium drying VGNKI-3 in the ratio 1:1 and lyophilizers. Determine the activity of the virus titration in cell cultures Fs before and after lyophilization and subsequent storage at a temperature of minus 20oWith an interval of six months. Lower caption herpesvirus drying on the environment VGNKI 0.5 lg TCD 50/cm3. Lower caption herpesvirus during storage for six months at a temperature of minus 20oWith 0.1 lg TCD 50/cm3in the year of 0.2 lg TCD 50/cm3, a half - 0,50,1 lg TCD 50/cm3two years 1,00,25 lg TCD 50/cm3.
3inactivate formalin to a final concentration of which is 0.2%. Five cats 4 months of age vaccine antigen in a volume of 1 cm3injected subcutaneously once. The maximum accumulation of antibodies in the blood sera of cats registered on the 21st day after its introduction. During this period, they were gradually increasing. The titers after a single injection of antigen in rnga is 5-6 log2.Example 6
Specific hyperimmune serum to strain herpesvirus cats "Grand" get on rabbits and goats. Use 2 methods of immunization: weekly 3 - fold introduction of antigen in a volume of 1 cm3rabbit and 3 cm3the goat and the immunization with low doses of antigen, where the antigen is injected intramuscularly in the same volume when adding complete adjuvant's adjuvant (1:1), and then after 2 months without adjuvant intravenously in a volume of 0.3 cm3and 0.5 cm3respectively. Selection of blood to obtain serum samples produced after 14, 21 and 28 days after the last injection of antigen.The activity of the obtained sera of ustaoset. The activity of the blood serum of rabbits received at weekly immunization, is 5.0 to 6.0 log2(PH), goats - 4,0-6,0 1og2. Higher activity in PH are the blood serum that are obtained from rabbits and goats immunized with low doses of antigen - 6,0-7,0 1og2and 7,0-8,0 1og2respectively. The obtained serum show no activity in the PH with the virus feline panleukopenia in cats. In rabbits and goats in the control group they are negative.Received hyperimmune serum used for serodiagnosis of infectious bovine rhinotracheitis and for specific treatment of diseased animals. Serum stored in lyophilized or native form at a temperature of minus 10-20oWith in a period of 24 months.Example 7
Get globulin of specific hyperimmune serum of the blood of goats with a titer of 6.0 log2in PH, and serum of cats with titers of 8.0 log2in PH. This is done using the conventional method of vysalivaniya a saturated solution of sodium sulfate.The precipitated globulin resuspending phosphate buffer solution pH of 7.2. Title globulin from the serum of the blood of goats after resuspendable is 8.5 log2in PH, serum cats 9,25 log2.
Diagnostic serum to the herpes virus cats, obtained as described in example 6, is used to identify isolates of the viruses isolated from cats with signs of respiratory disease. For this purpose the contents of the vial with lyophilized serum dissolved in 10 cm3the growth culture medium, warm up for half an hour at 56oC in a water bath. The analyzed samples isolates of herpesvirus and the control sample herpesvirus cats strains Grand (positive control antigen) is combined with the diluted serum in a 1:1 ratio. The resulting mixture is incubated for 1 hour at room temperature and contribute on 1 cm3in vitro culture of cells of the kidneys of cats on the formed monolayer (4 tubes for each sample). In parallel in 4 other tubes cell culture contribute on 1 cm3cultivation of isolates 1:2 in supporting culture medium without serum. Control culture cells are uninfected tubes with monolayer cells, which contribute on 1 cm3supporting culture medium without serum and virus. Everything PD isolates containing herpesvirus in cats, which is manifested by the absence of JRS in test tubes containing a mixture of virus and positive serum. In test tubes containing the same isolate herpesvirus without serum, JRS manifests itself in the first day of the characteristic rounding of the cells, and then their exclusion from the glass with formation of voids in the form of a "grid". In the study sample isolates that do not contain the herpes virus in test tubes with this isolate characteristic JRC is not shown, and in case of occurrence of nonspecific JRS it is also observed in the test tubes containing the isolate and antiherpesvirus serum. In the control tubes with an uninfected cell culture is not observed degenerative changes in the cell monolayer.Example 9
Assessing antigenic activity herpesvirus in the vaccine "Triket" against calicivirus, infectious bovine rhinotracheitis and feline panleukopenia in cats produced by Nobivac, Holland, ser. E. For this cats 2-4 months of age Vaccinium specified vaccine at a dose of 1 cm3according to the instruction for use. Before vaccination and 21 days in all vaccinated puppies take blood samples and get the serum.To determine the titer of neutralizing the Andes" in transplantable cell cultures Fs. As a positive control serum at PH use specific diagnostic serum to the herpes virus cats, obtained as described in example 6. Evaluation of reactions carried out after 7 days on the development of the JRC in cell culture. The titer of neutralizing antibodies in the blood sera of vaccinated cats is 1:16-1:128. When comparing the results of the titration of sera obtained from cats before vaccination and after, note that in all experimental animals, the titers of specific antibodies to the causative agent of infectious bovine rhinotracheitis increase not less than 2 times, and seronegative prior to vaccination of animals become not lower than 1:32.It was thus established that the herpes virus component in the vaccine "Triket" is active and, therefore, the vaccine for this component suitable for use.Example 10
The vaccine against infectious bovine rhinotracheitis in cats produced by cultivation herpesvirus in monolayer cells of the kidney kitten.For infection using culture herpesvirus pieces "Grand" level 2 passages with activity at PH 9 lg TCD 50/cm3in volume 1 cm3200 cm3supportive environment. Time of cultivation is 48-72 hours. the dice. To inactivate viral duties performed electron microscopy to prevent their contamination and determine contagious viral collection in cell culture. The activity of the virus in the PH must be at least 6 lg TCD 50/cm3. The virus inactivating formalin to a final concentration of which is 0.2%. Adjuvant add 3% solution of hidroxil in the amount of 10% by volume.Vaccine against infectious bovine rhinotracheitis in cats inactivated is a liquid pink or purple-pink color with loose sediment crashing when shaken in a homogeneous suspension. It is suitable for use within 18 months from the date of manufacture when kept at a temperature of 2-4oC.Prepared vaccine sterile fungal and bacterial relationships.Example 11
Obtained in example 10 vaccine against bovine rhinotracheitis in cats tested for antigenic activity.To do this 3-4-month-old seronegative kittens and rabbits weighing at least 3 kg vaccine injected subcutaneously with a single dose of 1.0 cm3. Serum of the animals at 21 days after immunization examined in PH. The title is 6-7 log2. The vaccine antigen.Example 13
Checking immunogenic properties of the vaccine against infectious bovine rhinotracheitis in cats, obtained in example 10. For this purpose, two groups of 4 seronegative kitten 3-4 months of age. The first group of kittens vaccine is injected subcutaneously once daily at a dose of 1.0 cm3. The second group of kittens (control) was injected in the same volume of culture medium without virus. After 21 days after the start of the experiment all kittens infect oronasal the koi herpes virus - strain "Grand activity of not less than 7 lg TCD 50/cm3that is administered in a dose of 1 cm3. Intact kittens control group ill 3 is inicom, conjunctivitis, pneumonia. The total clinical status of the vaccinated kittens in the observation period (21 days) remains normal, local reactions at the vaccine is not noted. Vaccine immunogen.Example 14
Study the effectiveness of vaccine produced according to example 10, in a clinical setting. This vaccine subjected to immunization 32 cats and 24 cat 19 purebred, 13 Persian, 10 Scottish fold, 9 British, 3 Siamese, 2 Devon Rex. The age of animals - from 2 months to 5 years, and 75% of the vaccinated animals under the age of 1 year. The vaccine is injected subcutaneously at a dose of 1 cm3.In any case, do not see post-vaccination reactions, both local and General. None of the animal is not noted oppression, loss of appetite, allergic reactions. Local skin reactions at the vaccine does not reveal. In any case, do not see post-vaccination complications and animal diseases infectious bovine rhinotracheitis during the year of observation.Example 15
Learn the medicinal properties of hyperimmune sera obtained in example 6, and globulins obtained in example 7. In the experience of use 45 cats of different breeds and age with a diagnosis of infectious bovine rhinotracheitis" same antibiotics nonspecific treatment complement the use of hyperimmune serum (obtained according to example 6) with a titer of 8.0 log2in 10 cases - globulin (obtained from example 7) from sera of goats, 11 - globulin from the serum of cats. Specific drugs administered to cats subcutaneously with 1.0 cm3a day for 3 days. In all cases, the use of specific drugs installing the recovery of cats, which occurs within 24 to 72 hours, which is earlier than usual, for 3-5 days, and in any case do not indicate recurrence or complications of the disease. In the group of cats, which shall only antibiotics, say at least 2 deaths disease; group, where are serotherapy, deaths don't celebrate. One kitten of breed Devon Rex registriert allergic reaction to the second introduction serum goats, manifested by redness of the skin and itching. This reaction removes a single introduction of a solution Dimedrol in conventional doses. With the introduction of globulins kittens of the same litter allergic reactions were noted. Studies establish therapeutic efficacy of the use of specific serum and globulin in the treatment of infectious bovine rhinotracheitis in cats.Use in veterinary practice strain of Feline herpesvirus-1, VGNKI "Grand"-31 allow sufficient do cats, to produce on the basis of a highly immunogenic vaccine against infectious bovine rhinotracheitis cats get hyperimmune sera of different animal species for the treatment and prevention of infectious bovine rhinotracheitis in cats. To use the antigen and received him in serum serological reactions for the diagnosis of infectious bovine rhinotracheitis in cats.Sources of information
1. Arentt B. D., Greene, C. E. Feline respiratory disease/The book: Clinical Microbiology and infectious diseases of the dog and cat (ed. Greene, C. E. ) Philadelphia. - 1984. - R. 527.2. Gillespie J. H. , and Scott F. W. Feline Calicivirus (FCV) infection. // Adv. vet. sci. and comp. med. - 1973. - 17. - p. 176-188. The strain of Feline herpesvirus-1 VGNKI "Grand"-31 used to control immunogenic vaccine manufacturer specific therapeutic and diagnostic Biologicals.
SUBSTANCE: it is necessary to carry out complex medicinal therapy including specific antiviral preparations, group B-vitamins, immunocorrectors, injection of antiherpetic vaccine, and, also local therapy with derinate. Additionally, since the 1st d of exacerbation it is important to inject flosteron once i/m at 1.0 ml, cefotaxym i/m twice daily per 1.0 mg for 10 d. Immunomodulator halavit on the 1st d should be prescribed i/m at the dosage of 200 mg, and then for the next 15 d - twice daily per 100 mg i/m. Diazepam for 1 mo should be injected per 5.0 mg 4 times daily, and then per 2.5 mg 4 times daily for 1 mo, as well. One should daily inject atarax per 100 mg nocturnally for 1 mo and ketorol internally per 10 mg thrice daily for 10 d. Moreover, it is necessary to apply the composition out of 50.0 g zinc oxide, 5.0 ml ACD fraction and 25.0 g "Aciclovir" cream 4 times daily for 10 d. The innovation enables to considerably increase the efficiency of therapy due to improving the main immunological values, weakening local autoimmune reactions and, also, preventing the development of psychic disorders in such patients due to affecting viral neurotropic properties.
EFFECT: higher efficiency of therapy.
3 dwg, 1 ex