RU2279474C1 - Strain of infection bovine rhinotracheitis (ibr) virus for production of vaccine and diagnostic preparations - Google Patents

Abstract

FIELD: veterinary virology and biotechnology.

SUBSTANCE: invention relates to new virus strain having high antigenic and immunogenic activity useful in preparation of inactivated vaccine against IBR.

EFFECT: IBR virus strain of increased antigenic and immunogenic activity.

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Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the manufacture of means for the specific prophylaxis and diagnosis of infectious rhinotracheitis (IRT) in cattle.

IRT is an acute contagious disease of cattle of viral etiology, causing significant economic damage to livestock.

IRT virus causes cattle disease of various age groups, affecting the respiratory (rhinotracheitis), genital (pustular vulvovaginitis, balanoprostitis) tracts, provoking abortions in cows, and also affects the nervous system (encephalomyelitis), mucous membranes and the eyeball in calves (conjunctivitis) . The disease is widespread throughout the world.

The economic damage caused by ИРТ consists of a decrease in milk yield during the period of illness (up to 50-60%), a significant increase in the service period and barbarity of cows with a vaginal form, poor development of sick young animals and a significant percentage of death and culling of calves.

Vaccine prophylaxis of IRT takes a leading place in the complex of antiepizootic measures aimed at combating this disease. For the prevention of IRT, both live and inactivated vaccines obtained from the virus reproduced in various cell cultures are used [1, 2, 3].

Although live attenuated strains are used to make live vaccines, they are nevertheless capable of causing complications on the immune background in animals. This may occur due to reactivation or recombination of the vaccine virus with the field. Epizootic strains are used in the manufacture of inactivated vaccines, but they can cause outbreaks in the case of incomplete inactivation of viral raw materials.

Known epizootic strain 4016 virus RTI of cattle, used for the manufacture of inactivated vaccines [4]. This strain is grown in labor-intensive primary trypsinized cell cultures of BT, PEC, PT with an infectious activity of 6.0-6.5 lg TCD ₅₀ / ml, which does not allow to obtain the required amount of antigen in a vaccination dose without prior concentration of the virus.

Known strain TK-A (VIEV) - In 2 virus IRT cattle, used for the manufacture of inactivated vaccines [5].

Known virulent strain of HERPES birds bovis - 1 "Schapovo" virus RTI of cattle, used for the manufacture of inactivated vaccines [6].

Known strain TK-A virus IRT cattle used for the manufacture of inactivated vaccines [7].

The closest is the virulent strain "ARRIAH" of the virus of infectious rhinotracheitis in cattle for the manufacture of vaccines and diagnostic preparations used for the manufacture of inactivated vaccines [8].

A common disadvantage of the known strains is the low antiepizootic efficacy of vaccines prepared on their basis. These strains have a low level of accumulation in sensitive cell systems, which does not allow to obtain the required amount of antigen in the vaccination dose to ensure reliable protection of animals from epizootic strains of the RTI virus.

The aim of the present invention was to obtain a new safe (attenuated) production strain of cattle IRT virus with high biological, antigenic and immunogenic activity, retaining its native immunobiological properties after inactivation and suitable for the manufacture of highly immunogenic vaccine preparations (from non-concentrated virus) that can protect cattle stock from epizootic causative agent of cattle IRT circulating in the Russian Federation.

The technical result from the use of the present invention is to expand the arsenal of safe (attenuated) strains of the RTI virus of cattle with high biological antigenic and immunogenic activity, retaining their native immunobiological properties after inactivation and suitable for the manufacture of highly effective vaccine preparations.

This technical result was achieved by obtaining a strain of TK-A / K virus IRT cattle. Strain TK-A / K is a new previously unknown. The original virus of strain TK-A was used to obtain the strain TK-A / K by cloning by limiting dilutions and multiple passages on a sensitive cell system.

The resulting strain was deposited in the All-Russian State Collection of microorganism strains used in veterinary medicine and livestock, the All-Russian Scientific Research Institute for Control, Standardization and Certification of Veterinary Medicines (VGNKI) of the Ministry of Agriculture of the Russian Federation on March 12, 2004 under the registration code TK-A / K - DEPT.

The strain TK-A / K of the IRT virus differs from the prototype in the uniformity of the population and higher biological, antigenic and immunogenic activity. The possibility of its use for the manufacture of diagnostic and prophylactic means for cattle IRT has been experimentally confirmed.

The strain TK-A / K provides for the serological diagnosis of cattle IRT and the production of an effective inactivated vaccine against cattle IRT, which provides reliable protection of cattle from the specified pathogen.

The strain TK-A / K of the IRT virus is characterized by the following features and properties.

The taxonomic characteristic is that the strain TK-A / K has the shape and dimensions typical of herpes viruses. In serological reactions (pH, RNGA, RSK, ELISA, etc.), antigens common to all strains of herpes virus of cattle are detected. The main marker characteristics of the strain TK-A / K of the IRT virus are shown in Table 1.

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

EXAMPLE 1

In order to obtain a genetically homogeneous population for isolation of a clone from the virus, ИРТ pcs. TK-A used the method of limiting dilution. The MDBK cell culture was infected with a final suspension diluted viral suspension and incubated at 36-37 ° C until the viral CPD appeared at the passage levels. As a result of prolonged passage, a new strain TK-A / K was obtained, which differs in genetic traits from its predecessor.

To obtain the results of the molecular genetic characteristics of the strain TK-A / K of the IRT virus, a restriction analysis of virion DNA isolated from preparations purified after selection was performed.

Upon cleavage of the DNA of the virus with various restriction enzymes, a set of fragments characteristic of each enzyme was obtained. Based on the obtained data, a restriction map was compiled, which characterizes the genetic structure of the DNA of the production strain of TK-A / K virus of IRT cattle.

The resulting virus was used for subsequent passage. Research results on the adaptation of the virus ИРТ pcs. TK-A / K in the MDBK cell culture are shown in Table 2.

The resulting virus was subjected to comprehensive control in accordance with the OIE guidelines for standard diagnostic methods and vaccines (1996) and at passage 6 after lyophilization, it was stored at minus 60 ° C as a production one with a titer of infectious activity of at least 6.5-7, 0 lg TCD ₅₀ / ml. The resulting strain of cattle IRT virus is assigned the author's name TK-A / K.

EXAMPLE 2

In order to determine the biological antigenic and immunological activity of the IRT virus of cattle, strain "TK-A / K" hyperimmunization of rabbits was carried out with live weight of 2.5-3.0 kg.

For hyperimmunization of rabbits, lyophilized virus ИРТ strain ТК-А / К was used. The contents of the ampoules were dissolved with saline to the original volume, then diluted 10 times by volume (5.5-6.0 lg TCD ₅₀ / ml.) The animals were injected with viral material from strain TK-A / K in a volume dose of 1.0 cm ³ intramuscularly three times with an interval of 14 days. 15 days after the end of hyperimmunization, the rabbits were bled, blood serum was obtained individually from each animal. Each serum was tested for activity and specificity in the neutralization reaction. The research results are given in table.3.

The data presented in Table 3 characterize the high biological, antigenic, and immunogenic activity of the strain TK-A / K of the RTI virus of cattle when introduced into the body of laboratory animals.

EXAMPLE 3

To obtain a sorbed inactivated vaccine against cattle ИРТ, the mother virus of strain TK-A / K is grown in a monolayer of MDBK cells by the roller method.

The production series of the IRT virus should have an infectious activity of at least 7.25-7.5 lg TCD5 ₅₀ / ml, activity in the RGA not less than 4.0 log ₂ .

Inactivation of viral raw materials is carried out using a 10% solution of theotropin, introduced into the suspension of the IRT virus to a final concentration of 0.1% dry matter. For this, a solution of theotropin is introduced into the virus-containing suspension with stirring, and the pH of the viral material is adjusted within 7.2-7.5 with a solution of potassium phosphate disubstituted. Inactivation is carried out at a temperature of 36-37 ° C for 7 days with periodic stirring (3-4 times a day for 2-3 minutes).

At the end of the inactivation process, the material is examined for completeness of inactivation in 3 consecutive passages in the MDBK cell culture. As the adjuvant sorbent in the vaccine, a sterile 3-6% colloidal solution of aluminum hydroxide (GOA) is used. The mixture of the suspension with GOA is stirred for 1 hour and left for sedimentation. Then, an additional 10% aqueous solution of saponin adjuvant is added to the semi-finished vaccine at the rate of 0.1% dry matter in final concentration. The mixture before packaging is stirred at a temperature of 10-12 ° C for 1 hour. The optimal component composition (wt.%) Of the obtained vaccine against cattle ИРТ is given in Table 4.

The content of antigen in the vaccine in the above range is its optimal amount in the drug, ensuring the achievement of a technical result.

The resulting vaccine is monitored for avirulence and sterility, and then packaged in sterile vials.

Sterility of the vaccine is determined in accordance with GOST 28085-89. The virulence of the drug is determined by the method of three consecutive passages of the inactivated semi-finished product in the transplanted MDBK cell culture by the absence of CPD.

The resulting vaccine is a pink liquid with a loose precipitate of the sorbent, which, when shaken, is easily broken into a homogeneous mixture.

Determination of the safety of the vaccine is carried out on mice or rabbits at a dose of 0.5 ml and 5 ml, respectively. A vaccine is considered harmless if laboratory animals remain clinically healthy for 10 days without any pathological manifestations. At the injection site, a local tissue reaction is allowed.

The antigenic activity of each vaccine series is tested in rabbits. The vaccine is administered twice with an interval of 14-28 days to four rabbits intramuscularly in the thigh area in a volume of 1 ml. 14 days after re-immunization, blood samples were taken from rabbits, incubated at 37 ° C, serum was separated and examined for the presence of antibodies to the IRT virus in pH or RNGA. The vaccine series is considered to have passed the control for antigenic activity if the titer of BH antibodies and antibodies in RNGA to the IRT virus is not lower than 1: 8 and 1:16, respectively.

The results shown in table 5, indicate that the inactivated vaccine against cattle IRT from strain TK-A / K has a high antigenic activity.

When studying the immunogenic activity of an inactivated sorbed vaccine against cattle ИРТ from strain ТК-А / К, it was found that in calves 30-40 days of age vaccinated twice subcutaneously in a dose of 2 ml already on day 14 after administration of the drug, an immune response is generated, providing 90- 100% formation of humoral antibodies and protection of animals from the disease. It has been experimentally shown that one vaccination dose in a volume of 2 ml contains at least 20 BMI ₅₀ . The research results presented in table 6, confirm the high efficiency of the inactivated vaccine against cattle IRT from strain TK-A / K.

Thus, the above information indicates the fulfillment when using the invention of the following set of conditions:

- strain TK-A / K virus IRT cattle, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology.

- for the proposed invention in the claims confirmed the possibility of its implementation using the methods and methods described in the application prior to the priority date.

- strain TK-A / K, obtained in accordance with the invention, has a high infectious, antigenic and immunogenic activity and is suitable for the manufacture of vaccines against IRT cattle.

LITERATURE

1. Infectious diseases of animals. Directory. Ed. D.F. Ozidze. - M .: Agropromizdat, 1987, 19-99.

2. Yurov K.P. and Shulyak A.F. Herpes viruses of causative agents of mass diseases of cattle. Veterinary Medicine, 1998, No. 11, 10-12.

3. Syurin V.N., Samoilenko A.Ya., Soloviev B.V. and Fomina N.V. Viral diseases of animals. - M., VNITIBP, 630-646.

4. Auth. testimonial. USSR No. 980307, A 61 K 39/12; 1981.

5. Auth. testimonial. USSR No. 1789219, A 61 K 39/118; 03/21/93.

6. Pat. RF №2059415, A 61 K 39/265, 05/10/96.

7. Pat. RF №2090210, А 61 К 39/265, С 12 N 5/06, 7/00, 09/20/97.

8. Pat. RF №2221040, А 61 К 39/265, С 12 N 7/00, 06/28/2002.

table 2
The results of the cloning of virus IRT cattle strain "TK-A / K" in the culture of MDVC cells Material for infection Passage level Dose of infection (TTZ _{50 / cl} ) The cultivation time (day) Virus titer lg TCD _{50 / ml} Original virus with 7.0 lg TCD _{50 /} cell 1st 0.0001 8 4.25 Passage 1 virus 2nd 0.0001 8 4,5 Passage 2 virus 3rd 0.0001 6 5,0 Passage 3 virus 4th 0.0001 5 5.75 Passage 4 virus 5th 0.01 four 7.0 5th passage virus 6th 0.01 3 7.5 Passage 6 virus 7th 1,0 2 8.0 7th passage virus 8th 1,0 2 7.75 Passage 8 virus 9th 1,0 2 8.25 Passage 9 virus 10th 1,0 2 8.0 Table 3
The level of accumulation of virus-neutralizing antibodies during hyperimmunization of rabbits with virus IRT cattle strain "TK-A / K" Rabbits number Serum antibody titer in pH 14 days after injection. Before introduction After the 1st injection After the 2nd injection After the 3rd injection one 0 1:16 1: 32-1: 64 1: 256-1: 512 2 0 1:16 1: 32-1: 64 1: 256-1: 512

Table 4
Prescription of the component composition (wt.%) Of the inactivated vaccine against cattle ИРТ prepared from strain "TK-A / K" Ingredients Minimum Average Maximum Antigenic material 60 70 80 Theotropin 0.1 0.1 0.1 GOA (6th gel) 39.0 29.0 19.0 10% saponin solution 1,0 1,0 1,0 Table 5
Antigenic activity of inactivated GOA-saponin vaccine against cattle ИРТ from strain "TK-A / K" No. p / p Name of material Antibody titer in the pH in rnga one Vaccinated rabbit serum 1:32 1: 128 2 -≪- 1:64 1: 256 3 -≪- 1:64 1: 128 four -≪- 1:32 1: 128 5 Serum of control (non-vaccine.) Rabbits 0 0

Table 6
Immunogenic activity of inactivated GOA-saponin vaccine against cattle IRT from strain "TK-A / K" №№ Name of test material Antibody titers in RNGA Before vaccination 15 days after the 1st vaccination 15 days after the 2nd vaccination one Vaccinated calf serum 1: 4 1:16 1: 128 2 - "- 1: 2 1: 8 1:64 3 - "- 1: 4 1:16 1: 128 four - "- 1: 4 1:32 1: 128 5 - "- 1: 2 1: 8 1:64 6 - "- 1: 8 1:32 1: 128 7 - "- 1: 4 1:16 1:64 8 - "- 1: 2 1: 8 1:64 9 - "- 1: 8 1:32 1: 256 10 - "- 1: 4 1:16 1: 128 eleven Serum control (unvaccinated) calves 1: 4 1: 4 1: 2

Claims (1)

Bovine Herpesviridae infectious rhinotracheitis virus strain, genus Varicello, subtype Herpesvirus bovis 1, VGNKI collection, “TK-A / K” - DEPT, for the manufacture of vaccines and diagnostic preparations.