RU2207372C1 - Strain "o" of virus of poultry infectious laryngotracheitis for preparing diagnostic and vaccine preparations - Google Patents

Abstract

FIELD: veterinary science, biotechnology, vaccines. SUBSTANCE: the parent virus for preparing strain "O" is isolated from larynx and trachea of chicken with symptoms of lethal form of sickness in Kazakhstan in 1992. The industrial strain "O" is obtained by multiple successive passages in 9-11 daily old embryos of SPF-chickens. The strain is deposited in Collection of microorganisms VGNKI 05. 06. 2000 at registration name "O = 112-DEP". The strain shows high biological, immunogenic activity both in native form and after inactivation. The strain is cultured in 9-11 daily old embryos of SPF- chickens and in 3-6 days of culturing the infectivity titer is 6.2 lg ЭИД₅₀/cm³, not less. The strain shows high biological, antigenic and immunogenic activity both in native form and after inactivation and useful for preparing highly specific and sensitive diagnostics and highly immunogenic, harmless and areactogenic vaccine preparations for ocular and oral methods of immunization. EFFECT: valuable properties of strain and preparations. 6 tbl

Description

 The invention relates to the field of veterinary virology and biotechnology and can be used in the development and production of diagnostic tools and specific prophylaxis of infectious laryngotracheitis (ILT) of birds.

 ILT, an acute contagious disease of birds, is the cause of a high mortality rate (usual mortality is 10-20%, there are cases of high mortality, which is 50-70%), in cases of ill individuals the daily gain and egg production are reduced. ILT is caused by the herpes virus. One of the main means of combating ILT is vaccination with live and inactivated vaccines.

 The advantages of the virus vaccines are that they are suitable for mass use by watering, aerosol spraying, etc., for the immunization of birds, a relatively small amount of the immunizing drug is required, since the live antigen continues to multiply in the body of the immunized bird, they are cheaper in production and for their storage, relatively little space is required in refrigeration units.

 In world practice, more than 20 strains of the bird ILT virus are known that are used for the manufacture of vaccine preparations (1-6). Known ILT virus strains differ in virulence for birds and chicken embryos (CE), tropism, release rate from cells in tissue culture, storage stability, avidity, molecular structural features, deviations of different virulence strains in the ability to neutralize hyperimmune sera (7-9 )

 The known strain "G" of the ILT virus used for the manufacture of vaccine preparations (10).

 Known strain "TSNIIPP" virus ILT birds used for the manufacture of vaccine preparations (3).

 Known clone "NT" strain "TSNIIPP" virus ILT birds used for the manufacture of vaccines (11).

 The disadvantages of the known strains are their low immunogenic activity. Vaccines from these strains form immunity, which is not able to protect 100% of the poultry head from infection with the epizootic ILT virus.

 The closest to the invention according to the set of essential features is the strain "VNIIIBP" virus ILT birds used for the manufacture of vaccines (12).

 The disadvantages of the prototype strain are its low immunogenic activity and increased reactogenicity.

 Given the large informational capacity of the ILT virus genome and the diversity of its phenotypic properties, the urgent problem is the search for new vaccine strains of this pathogen that have high biological, immunogenic and antigenic activity, both in native and inactivated forms, as well as low reactogenicity.

 The objective of the present invention was to expand the arsenal of ILT virus strains that have high biological, immunogenic and antigenic activity both in native and inactivated forms and are suitable for the manufacture of highly specific and sensitive diagnostics and highly immunogenic, harmless and areactogenic vaccines for ocular and oral methods immunization.

 The technical result from the use of the present invention is to expand the arsenal of strains of the ILT virus of birds with high biological, immunogenic and antigenic activity both in native and inactivated form and suitable for the manufacture of highly specific and sensitive diagnostics and highly immunogenic, harmless and aretogenic vaccines for ocular and oral methods of immunization.

 The indicated technical result was achieved by obtaining an attenuated strain "O" of the bird ILT virus. The strain is new, previously unknown. The initial virus for obtaining strain "O" was isolated from the larynx and trachea of chicken with signs of a latent form of the disease in Kazakhstan in 1992. Production strain "O" was obtained by repeated consecutive passages on 9-11-day-old embryos of SPF hens.

 Strain "O" was deposited in the All-Russian State Collection of strains of microorganisms used in veterinary medicine and animal husbandry, the All-Russian State Research Institute of Control, Standardization and Certification of Veterinary Medicines (VGNKI) 05.06.2000 under the registration name "O 112-DEPT".

 Strain "O" has a high biological, antigenic and immunogenic activity in its native form and after inactivation. The possibility of its use for the manufacture of diagnostic and vaccine preparations has been experimentally confirmed.

 Strain "O" virus ILT birds is characterized by the following features and properties.

Morphological properties
Strain "O" of the ILT virus belongs to the Herpesviridae family. Electron microscopy (instrumental magnification of 4.5 x 10 ⁵ and 5 x 10 ⁵ ) in the field of view of the microscope clearly visible viral particles of ILT with their characteristic morphology are observed. Virions have a spherical shape, their sizes vary from 200 to 250 nm (in some up to 330 nm). ILT virions consist of a spherical nucleocapsid surrounded by a complex-shaped supercapsid membrane. Virion has an icosahedral type of symmetry. The icosahedral capsid is covered with a lipoprotein membrane, which has on its surface morphological units-peplomeres. The diameter of the nucleocapsid is 113 ± 2 nm, the average diameter of the nucleocapsid membrane is about 350 nm.

 Nucleocapsids 100-110 nm in size are located in the viral particle, usually eccentric, less often in the center of the particles. The genome of the ILT virus has a length of 155,000 polynucleotides and consists of a long unique sequence of I1 (120,000 polynucleotides) and a short unique sequence of Is (17,000 polynucleotides). The virus contains glycoproteins (205, 160, 115, 90, 85, 74, 60 and 50 kD). Glycoprotein B (gB) is the first structural protein of the ILT virus.

Antigenic properties
Strain "O" of the ILT virus is stably neutralized by homologous serum, is identified in the blood serum of chickens containing antibodies to this virus, in RDP, PH and ELISA. In RDP, it is identified by homologous hyperimmune sera in a titer of 1: 8. In pH, the neutralization index of homologous hyperimmune sera at 1: 10 and 1:20 dilutions is on average 3.8 Ig EID ₅₀ / cm ^3, respectively. In ELISA is identified by field and laboratory hyperimmune sera in the titer up to 1: 24579. Antigenic differences of strain "O" in relation to other strains of the ILT virus have not been established. The strain does not have hemagglutinating properties.

Biotechnological characteristics
Strain "O" exhibits high biological, immunogenic and antigenic activity both in its native form and after inactivation. Strain "O" is used for the manufacture of live and inactivated vaccines against bird ILT and diagnostic preparations. The virus has a pronounced tropism to the epithelial cells of the mucous membrane of the larynx, trachea and cloaca of birds. The strain is cultured in 9-11 day old SPF chicken embryos. Reproduction of the virus occurs in the tissue of the chorioallantoic membrane (HAO). The infectious titer of the virus in a 10-20% suspension of HAO on the 5th day of cultivation under the condition of generalized distribution throughout the membrane is at least 6.2 Ig EID ₅₀ / cm ³ .

Chemo- and genotaxonomic characterization
Strain "O" is a DNA-containing virus.

Resistance to external factors
The virus is sensitive to lipolytic agents, heat and various disinfectants. A temperature of 55 ^o C destroys the virus within 10-15 minutes, and 38 ^o C - within 48 hours. In the frozen carcasses of sick and forcedly killed chickens stored at minus 10, 18 and 28 ^o C, the virus remained active for more than 19 months, on the surface of the shell in the conditions of a thermostat, it is inactivated after 12 hours, and in infected houses no more than 9 days remain. Fully inactivated in a 1% creole solution for 30 s. It retains its activity for a long time in a lyophilized state or at minus 20-60 ^o C. In the lyophilized homogenate of HAO, the virus remains infectious for at least 1.5 years. In the frozen tissue of HAO, the infectivity of the virus persists for at least 4 years.

The immunogenicity of the strain
When vaccinating chickens older than 15 days of age, the ocular method at a dose of 1000 EID ₅₀ and the oral method (drink) at a dose of 4000 EID ₅₀ induces the production of specific antibodies and creates immunity against ILT in 100% of vaccinated chickens. In the lyophilized HAO homogenate, the virus retains immunogenicity for at least 1.5 years.

Epizooticity of the strain
When vaccinated and intact birds were kept together, transmission of the vaccine virus of strain “O” by contact was not established.

Strain Maintenance Conditions
Lyophilized material from strain "O" is stored at a temperature of 4 ^o C, virus-containing HAO - at a temperature not exceeding minus 40 ^o C. For the lyophilization use the stabilizer "ARRIAH. For resuspension of the lyophilized virus, an isotonic phosphate-buffered solution of pH 7.4-7.6 is used.

Genetic purity
After 20 passages on the Khao 9-11-day-old embryos of SPF hens, no changes in the immunobiological properties of strain "O" were found.

Additional features and properties
Reactogenicity is absent.

 Pathogenicity is absent.

 Virulence is absent.

 Oncogenicity is absent.

 There is no contagiousness.

 The attenuation stability was observed during 4 passages on sensitive chickens.

 Based on the data obtained, it can be argued that the strain "O" of the ILT virus according to antigenic and immunological spectra is an original, taxonomically new, previously unknown variant of the ILT virus.

 According to the applicant, the proposed strain meets the conditions of patentability "novelty" and "inventive step".

 The essence of the invention is illustrated by examples of its use.

 EXAMPLE 1

 ILT virus seed brood on SPF chicken embryos is prepared as follows.

A hole with a diameter of 8-10 mm is formed in the shell of the chicken embryo from the side of the air chamber. Four punctures in the corners of the shell and HAO with an injection needle are made into the corners of an imaginary square with a diagonal of about 5 mm, after which a virus-containing material from strain O is applied in a volume of 0.1 cm ³ . The hole in the shell is sealed with adhesive tape. Infected embryos are incubated for 5 days, and on day 6, the characteristic lesions of ILO are detected. A 15-30% suspension is prepared from the affected HAO, which is frozen and freed from coarse particles by low-speed centrifugation. The resulting virus-containing liquid with a biological activity of not less than 6.0 Ig EID ₅₀ / cm ^{3 is} used as a matro brood for mass infection of 9-11-day-old SPF-chicken embryos.

 EXAMPLE 2

 The vaccine virus against bird ILT from attenuated strain "O" is prepared as follows.

At the first stage, matte material is prepared from the production strain “O” of bird ILT virus as described in Example 1. A virus-containing suspension obtained from diseased HAO is used for mass infection of 9-11-day-old SPF chicken embryos. Infection of embryos is done through a puncture through the air chamber under the CAO into the allantoic cavity to a depth of 3-6 mm. The volume of the inoculum is 0.2 cm ³ and incubated for 3-6 days at 37.7 ^o C and a humidity of 60%. According to the results of ovoscopy and subsequent autopsy, the presence of HAO lesions characteristic of the ILT virus is determined. Affected HAO is used as a virus-containing material. A 15-30% suspension is prepared from the affected HAO, which is frozen and freed from coarse particles by low-speed centrifugation. The virus-containing supernatant is carefully drained under sterile conditions, and then a stabilizer is added to it, which can be used (separately or in combination) skim milk, lactose, gelatose, sucrose, etc. The virus-containing liquid and stabilizer are mixed in a ratio of at least 17: 3-19: 1. The resulting mixture is packaged in bottles, frozen at a temperature of minus 60-80 ^o C and subjected to lyophilization. The lyophilized material is sealed under vacuum and subjected to immunological activity testing with ocular and oral methods of vaccination at 1000 and 4000 EID _50, respectively, and for harmlessness through ocular and oral methods of inoculation at 10000 and 40,000 EID _50, respectively.

The resulting vaccine, when titrated on 10-day-old embryos of SPF chickens, has a titer of 6.2 to 6.6 Ig EID ₅₀ / cm ³ .

 The resulting vaccine is a lyophilized homogenate of virus-containing HAO embryos of SPF-hens, which has the form of a homogeneous finely porous beige structure in the form of a cylindrical tablet, soluble in water or a special solvent.

 Before use, the virus vaccine is diluted with a diluent, which is a 5% solution of glycerol in a phosphate-buffered solution, labeled with a neutral food coloring such as indigo carmine, and is intended for the preparation of a working dilution of the vaccine with the ocular method of immunizing birds and adjusting the pH of water to neutral or slightly alkaline, with the oral method of immunization.

 A clinically healthy bird older than 15 days old must be vaccinated. Birds up to 60 days of age are vaccinated twice with an interval of 14 days. Immunity is formed 7 days after revaccination. Revaccination is carried out every 6 months.

 EXAMPLE 3

Tests of the immunogenic activity of the virus vaccine against bird ILT, made as described in example 2. Determination of the immunogenic activity of the vaccine was carried out on chickens. For this, vaccine dilutions on a diluent were preliminarily prepared: for the ocular immunization method so that 0.05 cm ³ (the volume of one drop) contained 1000 EID ₅₀ , for the oral one, that 4000 EID _{50 were} contained in 1 cm ³ . In the experiment, 50 chickens were taken and 20 of them were immunized ocularly (1 drop per eye), 20 orally (they were fed 1 cm ³ ), and 10 chickens were left for control. Eighteen days after vaccination, the experimental and control chickens were infected with the virulent Bogatischevsky strain of avian ILT virus. The virus was administered intratracheally at a dose of 500 EID ₅₀ in a volume of 0.5 cm ³ . Chickens were observed for 10 days. A vaccine is considered immunogenic if it protects 16 chickens out of 20 vaccinated against ILT disease. In the control group, at least 8 chickens out of 10 infected should be ill with signs of ILT. The test results are shown in table 1, which indicate that all three prepared series of virus vaccines against ILT birds from strain "O" for all the tested parameters meet the requirements laid down in the technical conditions for the drug and are almost equally effective for ocular and oral use in birds . In the control groups of chickens, the incidence of ILT was 100%, while the mortality rate in 3 experiments was 48.6%.

 EXAMPLE 4

 An inactivated ILT vaccine is prepared from strain O of a virus grown in developing SPF chicken embryos as described in Example 2. The resulting virus is inactivated with aminoethylethylenimine and sorbed onto Aerosil A-300 with the addition of saponin. The results of studies to determine the immunogenic activity of inactivated vaccines from strain "O" are presented in table. 2.

 EXAMPLE 5

 Diagnostic serum is prepared as follows. SPF chickens 25-30 days of age are inoculated with an oral or ocular live vaccine from strain "O" of the ILT virus. After 14 days, all chickens are injected with an intramuscularly inactivated antigen with an oil adjuvant, and after two weeks the chickens are bled and hyperimmune active serum is obtained and its activity in RDP and ELISA is determined. The research results are presented in table. 3.

 EXAMPLE 6

 Diagnostic serum is prepared as follows. SPF chickens 25-30 days of age are given an oral or ocular live vaccine from strain "O" of the ILT virus. After 14 days, intramuscularly inactivated antigen was inoculated to all chickens using hydrophobic aerosil or alumina hydrate as adjuvant. After two weeks, the chickens are bled, receive serum and determine its activity in the RDP and ELISA. The research results are presented in table. 4.

 EXAMPLE 7

 Dry diagnostic serum is prepared as follows. SPF chickens 25-30 days of age are given an oral live vaccine from strain O of the ILT virus. After 21 days, intramuscularly inactivated antigen with oil adjuvant was inoculated to all chickens. After 7 days, the inactivated antigen is reintroduced and after 7 days the chickens are bled, receive serum and determine its activity. Stabilizing additives are added to the obtained serum and it is lyophilized. Lyophilized serum is used for ELISA. The results of serum studies are presented in table. 5.

 EXAMPLE 8

 A comparative assessment of the immunogenic activity of virus vaccines against ILT of birds prepared from strains of domestic and foreign origin: the Laryngo-vac virus vaccine (Holland), the virus vaccine from the NT clone of the TsNIIPP strain, and the virus vaccine from the strain "VNIIBP" and the vaccine virus from strain "O". A study of the effectiveness of these vaccines was carried out on SPF chickens of 25 days of age. One dose of test vaccine was administered to the chickens orally and ocularly. Evaluation of the effectiveness of vaccines was carried out according to the results of the control of infection with the virulent strain "Bogatishchevsky" virus ILT. The results of the studies are presented in table. 6.

 The data table. 6 indicate that during the control infection of vaccinated chickens with the virulent strain "Bogatishchevsky" of the ILT virus, only the vaccine virus from strain "O" induced the formation of immunity to ILT in 100% of vaccinated chickens.

Thus, the above information indicates the fulfillment when using the invention of the following set of conditions:
- strain "O" of the ILT virus, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology;
- strain "O" of the ILT virus, obtained in accordance with the invention, has a high biological, antigenic and immunogenic activity in its native form and after inactivation and is suitable for the manufacture of diagnostic and vaccine preparations;
- for the proposed invention in the form described in the independent claim, the possibility of its implementation using the means and methods described in the application or known prior to the priority date is confirmed.

 Therefore, the present invention meets the condition of patentability "industrial applicability".

 Sources of information taken into account when drawing up the description of the invention to the application for the grant of a patent of the Russian Federation for the invention of the "Strain" O "ILT virus of birds for the manufacture of diagnostic and vaccine preparations."

Sources
1. US patent 3331736; 167-78, 07/18/67

 2. US patent 3444293; 167-78, 05/13/69

 3. Auth. testimonial. USSR 129791; 30h, 6; 1960 year

 4. Auth. testimonial. USSR 1750683, A 61 K 39/245, 07/30/92

 5. RF patent 2138290, A 61 K 39/245, 39/265, 35/78; September 27, 1999

 6. Syurin V.N., Samuilenko A.Ya. and other viral diseases of animals. M., VNITIBP, 1998, 672-683.

 7. Glissen J.R. Proceedings, 1988, 137.

 8. Goodwin M.A. et al. Avian Diseases, 1995, 39, 2,444.

 9. Guo Peixuan et al. Virology, 1944, 202, 2, 771.

 10. Babkin V.F. et al. Veterinary medicine. Rep. mizhvid. thematic sciences. 3b. Kiev, 1975, no. 42.14-19.

 11. Dutko Yu.S., Shevchenko A.A. and others. Immunological efficacy of a virus vaccine against infectious laryngotracheitis of hens. Veterinary Medicine, 1991, 3, 32-34.

 12. Malushko V.V., Sokolov V.D. et al. Experience in combating infectious laryngotracheitis. Poultry farming, 1983, 12, 21-23 (prototype).

Claims (1)

 The strain of the virus of infectious laryngotracheitis of birds, this. Herpesviridae, genus Herpesvirus, collection of VGNKI "About 112-DEPT" for the manufacture of diagnostic and vaccine preparations.

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