RU2266327C1 - Transmissible gastroenteritis of swine virus strain for production of diagnosis and vaccine preparations - Google Patents

Abstract

FIELD: veterinary virology and biotechnology.

SUBSTANCE: transmissible gastroenteritis of swine virus strain (TGSV) was isolated by sequential passages on SPEV (swine embryo kidney cell) and other cell cultures. Said virus strain is most effectively cultivated in 3-4-day monolayer of SPEV cells, obtained in roll-tube vessel, wherein after cultivation for 24-30 h infective activity level is 7.5 lg TCD₅₀/cm³. Strain survives starting characteristics for 18-month storage and for 9-10 passages (observation time) on SPEV cell culture.

EFFECT: TGSV strain with high biological, antigenic and immunogenic activity useful in production of high-specific and sensitive diagnosticums and high-immunogenic and harmless inactivated vaccine preparations.

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Description

The strain "Krasnodon" virus of transmissible gastroenteritis of pigs for the manufacture of diagnostic and vaccine preparations.

The invention relates to the field of veterinary virology and biotechnology and can be used in the manufacture of diagnostic and prophylactic means for transmissible gastroenteritis of pigs (THES).

Transmissible gastroenteritis of pigs (infectious gastroenteritis of pigs, Doyle and Hutchings disease) is an infectious highly contagious disease, mainly piglets up to 3 months of age, characterized by catarrhal hemorrhagic gastroenteritis, vomiting, profuse diarrhea, dehydration of the body and accompanied by high intestinal atrophy and tuberculosis mortality (often up to 100%) among piglets up to 2 weeks of age.

The disease is widespread in most countries of the world with developed pig husbandry, including in the CIS countries, and causes very significant economic damage. It consists of the high mortality of newborn piglets, the low effectiveness of prevention and treatment, as well as the difficulty in preventing the introduction of infection.

The causative agent of THES has a viral etiology. Its viral etiology was confirmed by American researchers Doyle and Hutchings (1946), when they described the filterability of the etiological agent TSHP. The TSHV virus was first isolated and described by the Japanese researcher Taiama (1970). The THES virus belongs to the Coronaviridae family, the genus Coronavirus.

To date, in various regions of the world a large number of isolates of the HHV virus have been isolated that do not have serotypic differences, and according to the results of the neutralization reaction (PH), they are assigned to the same serotype. The isolated isolates cause a similar clinical picture when infected susceptible piglets 1-10 days old obtained from sows non-immune to the THES virus.

Isolated isolates of the HHV virus are used to produce vaccines, the effect of which is based on the fact that the neutralizing colostral antibodies produced by the sows in response to the introduction of the vaccine virus protect the suckling pigs from infection with the virulent HHV virus.

For the prevention of HHP in Russia and abroad, live and inactivated vaccines from attenuated or killed HHP virus of various strains have spread (1).

Known strain V-52 of the THES virus for the manufacture of vaccine preparations (2).

Known strain "No. 5 VGNKI" virus TPP for the manufacture of vaccine preparations (3).

The known strain "MK-25" of the TGES virus for the manufacture of vaccine preparations (4).

The known strain "onion" of porcine respiratory coronavirus (RKVS) for the manufacture of vaccines against THES (5).

Known strain CAPM-V355 RKVS for the manufacture of vaccines against THES (6).

The closest to the invention according to the set of essential features is the strain "No. 1941 VNIIVViM" of the TGES virus for the manufacture of vaccine preparations (7).

The disadvantages of the known strains, including the strain of the prototype, are:

1) in their residual virulence and low immunogenic activity;

2) unsuitable for the manufacture of inactivated vaccines.

The task of creating the present invention was to obtain a new production strain of the TGES virus, which has high biological, antigenic and immunogenic activity in its native form and after inactivation and ensures the production of highly specific and sensitive diagnostics and highly immunogenic and harmless inactivated vaccine preparations.

The technical result from the use of the present invention is to expand the arsenal of production strains of the THES virus with high biological, antigenic and immunogenic activity and suitable for the manufacture of highly specific and sensitive diagnostics and highly immunogenic and harmless inactivated vaccine preparations.

The specified technical result was achieved by obtaining the strain "Krasnodonsky" of the TGES virus (copyright).

The strain Krasnodonsky was isolated in 1998 from the small intestine of sick sucking piglets belonging to ZAO Krasnodonskoye in the Volgograd Region with serial passages on the primary culture of pig kidney cells (SP) and adapted at passage 28 to the transplanted monolayer culture of pig embryonic kidney cells (SPEV) ) with a titer of at least 7.5 Ig TCD ₅₀ / cm ³ . The virus strain "Krasnodonsky" is avirulent for pigs.

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 April 27, 2001 under the registration name "attenuated epitheliotropic culture strain" Krasnodonsky - VNIIZZH- DEP "TGES virus.

Compared with the prototype, the strain "Krasnodonsky-ARRIAH-DEPT" of the TGES virus has a higher biological, antigenic and immunogenic activity in its native form and after inactivation. The possibility of its use for the manufacture of diagnostic preparations and an inactivated vaccine against HHPP, which protects susceptible animals against the pathogen circulating in Russia, has been experimentally confirmed.

The strain "Krasnodon ARRIAH-DEPT" virus TPP is characterized by the following features and properties.

Morphological properties

An electron microscopic study of the virus-containing material revealed that the virus of the Krasnodonsky-VNIIZZH-DEP strain is shell, pleomorphic, with a diameter of 80-150 nm, has one layer of club-shaped processes on the projection surface 12-25 nm long, i.e. the strain is morphologically identical to the representatives of this. Coronaviridae, genus Coronavirus.

Antigenic properties

The strain "Krasnodonsky-ARRIAH-DEPT" of the THES virus induces the synthesis of neutralizing and precipitating antibodies to the THES virus in the body of pigs and rabbits. Its antigenic affinity was studied in a cross-reaction of microneutralization (RMN). The virus does not show hemagglutinating activity (GA activity).

Biotechnological characteristics

The strain "Krasnodonsky-ARRIAH-DEPT" is attenuated, epithelial, cultural. Most efficiently propagated in a transplanted cell culture SPEV with a pronounced CPD, manifested in rounding and cell death, followed by destruction of the monolayer 24-30 hours after infection.

The optimal conditions for the growth of the THES virus in the SPEV cell culture are infection of a 3-4-day monolayer obtained on roller vessels at a dose of 0.1 TCD ₅₀ / cell and harvesting 24-30 hours after infection.

The virus titer in the specified cell culture reaches 7.5 Ig TCD ₅₀ / cm ³ . When titrating a viral suspension, it was found that at 70-80% CPD, the virus titer is 0.5-1.25 Ig TCD ₅₀ / cm ³ higher than at 90-100% monolayer damage. The results of adaptation of the proposed strain to transplantable cell cultures, studying the dynamics of virus accumulation in the cell culture of SPEV depending on the age of the monolayer, the time of cultivation and the dose of infection of the cell culture with the virus are shown in tables 1, 2, 3 and 4.

Chemo- and genotaxonomic characterization

The strain "Krasnodonsky-ARRIAH-DEPT" is an RNA-containing virus. The virus contains one large polyadenylated plus RNA (> 23 kDa). RNA extracted from virions is infectious. The virus was examined by PCR, and only genomic RNA was confirmed in the sample.

In the process of virus replication, 5-7 subgenomic mRNAs are synthesized with common 3'-ends for the production of viral proteins. The nucleotide sequence at the 3'-end of the genome corresponding to mRNA was determined. The floating density of the virions in the sucrose gradient is 1.18-1.20 g / cm ³ . Phospho- and glycolipids incorporated into the membrane are of cellular origin, i.e. the virion membrane is cell-dependent. The virus contains 3 major structural proteins: nucleocapsid protein (N), small membrane glycoprotein (M), and large peplomer glycoprotein (S).

In the mature virion, the N-protein is "woven" into the viral RNA, forming a ribonucleoprotein complex.

The heterophilic N-terminus of glycoprotein M protrudes from the virion, has the only available glycoprotein site and provides the induction of complement-binding and virus-neutralizing antibodies, as well as interferon. Glycoprotein S (or peplomeric protein) molar mass of 195-220 kDa looks on electron diffraction patterns as a corona of the virus. He is responsible for the attachment of the virus to the cell, the melting of the membrane and the induction of complement-independent virus neutralizing antibodies. Purified S-glycoprotein induces the formation of virus-neutralizing antibodies that neutralize virus replication at all stages. Defined 3 'portion of the genome of the virus. This region contains 3 major structural genes with 5'-S-M-N-3 'coronavirus orientation matching and 4 large open reading frames that encode potential viral proteins.

5 'part of the genome encodes a viral replicase / transcriptase.

Virulent properties

The strain "Krasnodonsky-VNIIZZH-DEP" is avirulent for pigs.

Immunogenic properties

When introduced into laboratory animals (rabbits, guinea pigs) and suckling pigs, it causes the formation of specific antibodies and the resistance of suckling piglets to direct infection with virulent strains of the THES virus.

Resistance to external factors

The virus of the strain "Krasnodonsky-VNIIZZH-DEP" is sensitive to ether, chloroform and sodium deoxycholate, resistant to trypsin. It is not suppressed by DNA inhibitors. Inactivated by 0.03% formalin solution, 1% Lysovet (a mixture of phenol and aldehyde), 0.01% solution of β-propiolactone, sodium hypochlorite, sodium hydroxide, iodine. The virus is resistant to freezing. Virus-containing material remains frozen for 5-8 weeks, at -18 ° C for up to 18 months, at -20, -40 or -80 ° C for at least 365 days. When heated to 56 ° C, n is activated in 30 minutes and up to 50 ° C in the presence of 1 M MgCl ₂ - in 1 hour. At 37 ° C, a complete loss of infectivity occurred after 4 days. Resistant to pH (4-9). Amantadine and puromycin inhibit the reproduction of the virus. The virus is stable in the bile of piglets at pH 3.0, preserving its infectivity in the small intestine.

Storage conditions

The strain is stored in the native state at a temperature of -70 ° C, pH of 7.2-7.4. Permissible storage period without refreshment is 18 months.

The stability of the basic properties of the strain

Within 18 months of storage, it does not reduce the titer of infectious activity, preserves antigenic (in RMN) and immunogenic activity, as well as the stability of genetic properties both during storage and in passages on SPEV (9-10 passages).

Additional strain information

Free from contamination by bacteria, mycoplasmas, fungi and other viruses.

Based on the data obtained, it can be argued that the strain "Krasnodonsky-VNIIZZH-DEP" on the antigenic and immunogenic spectra is an original, taxonomically new, previously unknown isolate of the TGES virus.

According to the applicant, the proposed strain meets the patentability conditions “novelty” and “inventive step” established by the Patent Law of Russia.

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

Example 1

The original virus for obtaining the strain Krasnodonsky-VNIIZZH-DEPT was isolated in 1998 from the small intestine of sick sucking piglets belonging to ZAO Krasnodonskoye in the Volgograd Region using serial passages on the primary cell culture of the joint venture.

The vaccine strain was obtained at FSI ARRIAH by adapting to the transplanted cell culture of SPEV. The strain is avirulent for pigs.

The results of studies on the adaptation of the TGES virus of the strain "Krasnodonsky-ARRIAH-DEPT" to transplantable cell cultures are presented in table 1.

The resulting virus was subjected to comprehensive control in accordance with the OIE guidelines for standard diagnostic methods and vaccines (1996) and at passage level 28 in the SPEV cell culture was stored as a mother seed. The virus of seed brood with a titer of infectious activity of 7.5 Ig TCD ₅₀ / cm ³ is presented in the form of a native suspension of culture fluid and the affected monolayer, stored at a temperature of -70 ° C.

The resulting strain of the TGES virus is assigned the copyright name "Krasnodonsky". The strain is deposited in the collection of microorganisms VGNKI under the registration name "attenuated epitheliotropic culture strain" Krasnodonsky-ARRIAH-DEPT "virus TGES.

Example 2

The virulent properties of the strain "Krasnodonsky-VNIIZZH-DEPT" were studied on "domolosive" pigs.

For comparison, the experiments also used a suspension of "minced meat" used in farms for the manufacture of a "tissue" vaccine for immunizing sows against THES, and attenuated strains of Purdue (reference) and No. 5 VGNKI. The latter is used for the production of virus vaccines at the Omsk Biocombine.

Each infected and control group of animals was kept in isolation. Clinical observations were conducted for 48 hours. The results of these experiments are shown in table 5.

According to table 5, the strain Krasnodonsky-VNIIZZH-DEP, as well as the strains Purdue and No. 5 VGNKI, are avirulent, since all infected pigs during the observation period remained clinically healthy. All piglets infected with a suspension of "minced meat" became ill after 18 hours with a characteristic clinical picture of THES. Control, i.e. uninfected piglets remained clinically healthy for 48 hours (observation period).

Example 3

Comparative tests of the antigenic properties of strains Krasnodonsky-VNIIZZH-DEPT and Purdue of the TGES virus were carried out. For this, hyperimmune sera from adult rabbits were obtained on them. In the experiments, purified antigens with an infectious activity of at least 7.5 Ig TCD ₅₀ / cm ^{3 were used} , which were mixed with Montanide ISA-70 oil adjuvant in a ratio of 1: 1.5 to obtain a water-in-oil emulsion. The resulting preparations were administered to rabbits intramuscularly in a volume of 2.0 cm ³ twice with an interval of 14 days. Blood samples from rabbits were taken before and 14-30 days after immunization, and serum was examined at the Russian Medical Center for the presence of antibodies against the reference Miller strain of the THES virus. The research results are presented in table 6.

From the data presented in table 6, it can be seen that on the 14th day after immunization against test strains, specific virus-neutralizing antibodies were formed in rabbit blood serum. After re-immunization, the antibody level in all groups increased, while the average antibody level in the group of animals immunized with an emulsion preparation from the Krasnodonsky-VNIIZH-DEP strain was 5.25 ± 0.25 log ₂ , which turned out to be slightly lower than the average antibody level 6.66 ± 0.31 log ₂ in the group of animals immunized with an emulsion preparation from the Purdue strain.

Thus, as a result of the studies, it was found that the proposed strain has high antigenic activity and is suitable for the manufacture of diagnostic drugs.

Example 4

An inactivated emulsion vaccine against HHPP from the strain "Krasnodonsky-VNIIZZH-DEPT" is obtained as follows.

For the manufacture of the vaccine, a culture of SPEV cells grown in roller vessels is used with a good monolayer of 3-4 days of age. As a growth medium, PSP medium with the addition of serum is used. From 90 to 100 roller vessels with a cell culture of 3.0 dm ³ each are taken to make 20 liters of a virus-containing suspension of the TGES virus. After the growth medium is drained, the cell monolayer is washed with a Hanks solution or phosphate-buffered solution with a pH of 7.2-7.4, then 3-5 cm ³ of matrix-type THES virus are introduced into the roller vessels. After 40-60 minutes of incubation at a temperature of 37 ± 2 ° C, 250-300 cm ^{3 of} support medium (without cattle serum) are introduced into the roller vessels.

Infected roller vessels are placed in a thermal room on rotary roller plants for cultivation at 37 ± 2 ° C.

The cultivation period is from 24 to 28 hours. For control, 2 roller vessels are left uninfected, in which the growth medium is changed to 300 cm ^{3 of} support medium. Starting at 18 hours of incubation, infected and control roller vessels are periodically microscopic. Roller vessels in which cytopathic changes are observed with damage to up to 70% of the cell monolayer (usually after 24-28 hours) are collected and 3 times frozen at -20 ° C and thawed. After that, the contents of the roller vessels are poured into one container and the cooled virus-containing suspension, subject to sterile conditions, is freed from cell detritus by centrifugation at 3000 rpm for 20 minutes. The suspension purified from detritus has the appearance of a clear pinkish-cherry liquid. Gentamicin (50 μg / cm ³ ) was added to the purified suspension and stirred for 20 minutes. Then, a sample is taken from the container for laboratory production control on the sterility and infectious activity of the virus. The production series of THES should have an infectious activity of at least 6.5 Ig TCD ₅₀ / cm ³ . The resulting virus is subjected to inactivation. For this, a 25-32% solution of aminoethylethyleneimine (AEEI) is added to a virus-containing suspension heated to 26-28 ° C with constant stirring to a final concentration of 0.008% and the pH value is set to 7.2-7.6. Inactivation is carried out at 37 ± 2 ° C for 22-24 hours with periodic stirring. At the end of inactivation, the suspension is cooled to 4-6 ° C, maintaining the pH at 7.2-7.6. Samples of antigenic material are then taken to test for sterility and avirulence.

An antigen from the TGES culture virus is considered suitable for the preparation of a vaccine if:

- there is no fungal-bacterial microflora;

- TGES antigen at SPEV does not cause a CPP.

An emulsion vaccine is prepared by dispersing a mixture of antigenic material with Montanide ISA-70 oil adjuvant (Seppic, France). For this, 2 parts of antigen and 3 parts of oil adjuvant are passed through colloid mills for 5-10 minutes and the prepared inactivated emulsion vaccine is collected in one sterile container. The vaccine is packaged in 10, 50, 100 and 200 cm ³ in sterile glass bottles, which are closed with sterile rubber stoppers and rolled up with aluminum caps.

The finished vaccine is an emulsion of white-pink color. When storing the vaccine, a white or pinkish-white loose sediment may form, eliminating by shaking.

The resulting vaccine is subjected to control on the content of hydrogen ions (pH) in the range of 7.3 ± 0.3, sterility, stability, viscosity (55 ± 20 mm ² / s), complete inactivation, safety and immunogenic activity.

The vaccine is intended for prophylactic immunization of pigs against THES.

Example 5

Tests of the immunogenic activity of the inactivated emulsion vaccine against HHPP from the strain "Krasnodonsky-VNIIZZH-DEP", manufactured as described in example 1.

Evaluation was carried out according to the results of a study in the Russian Medical Science against the serum and colostrum TGES strain No. 5 VNGKI from vaccinated sows and piglets obtained from them. The results of these studies are shown in table 7.

Table 7 presents compelling evidence that the proposed strain has high immunogenic activity, harmlessness and is suitable for the manufacture of vaccine preparations.

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

- strain "Krasnodonsky-VNIIZZH-DEP", embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology;

- for the proposed invention in the form as 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;

- strain Krasnodonsky-VNIIZZH-DEP, obtained in accordance with the invention, has high biological, antigenic and immunogenic activity, is suitable for the manufacture of highly specific and sensitive diagnostics and highly immunogenic and harmless vaccine preparations and expands the arsenal of new production strains of the TGES virus.

Therefore, the proposed strain meets the condition of patentability "industrial applicability".

Sources of information

1. Syurin V.N., Samuilenko A.Ya., Soloviev B.V. and other viral diseases of animals. - M.: VNITIBP, 1998 .-- S.165-183.

2. US patent No. 4046875, 424-89, 1977.

3. Autosvid. USSR No. 734279; C 12 N 7/00, 5/00; 05/15/80.

4. Autosvid. USSR No. 1238294, A 61 K 39/225, 10.20.95.

5. Sergeev V.A., Rukhadze G.G. and Alipper T.I. Pig respiratory coronavirus. Bulletin of S.-kh. Science, 1989, 8 (396). - S.96-100.

6. Autosvid. USSR No. 1731812, C 12 N 7/00; 05/07/92.

7. RF patent No. 2140982; C 12 N 7/00, A 61 K 39/255; 11/10/99 (prototype).

Table 1 The results of the adaptation of the epizootic isolate "Krasnodonsky" of the THES virus to transplantable cell cultures Isolate Cell culture No. of passages The titer of the virus, Ig TCD ₅₀ / cm ³ PPK nine 6.25 ± 0.12 PSGK-30 7 5.0 ± 0.10 "Krasnodonsky" RK-15 6 5.5 ± 0.5 3 2.5 ± 0.25 SPEV 5
12 4.0 ± 0.5
7.0 ± 0.15 28 7.5 ± 0.5 table 2 The results of the accumulation of the TGES virus of the strain "Krasnodonsky-VNIIZZH-DEP" in the cell culture SPEV depending on the age of the monolayer The age of the monolayer, (day) The titer of the virus, Ig TCD ₅₀ / cm ³ 2 6.5 ± 0.25 3 7.5 ± 0.15 4 7.5 ± 0.25 5 6.15 ± 0.15 Table 3 The results of the study of the dynamics of the accumulation of the TGES virus of the strain "Krasnodonsky-ARRIAH-DEPT" in the cell culture SPEV Cultivation time The titer of the virus, Ig TCD ₅₀ / cm ³ CPP,% 24 7.0 ± 0.25 70 26 7.5 ± 0.33 75 28 7.5 ± 0.25 75 thirty 7.5 ± 0.15 90 32 6.75 ± 0.25 100 44 6.25 ± 0.15 100

Table 4 The results of the study of the effect of the infection dose and the cultivation time on the accumulation level of the THES virus of the Krasnodonsky-VNIIZZH-DEP strain in the SPEV cell culture Experience Dose of infection, TCD ₅₀ / cl The time of reproduction of the virus (with a CPD of 70-80%), hours The level of accumulation of the virus, Ig TCD ₅₀ / CL 1 1,0 27 6.25 ± 0.15 0.1 thirty 7.5 ± 0.15 0.01 36 6.75 ± 0.25 2 1,0 27 6.5 ± 0.15 0.1 29th 7.5 ± 0.25 0.01 36 6.25 ± 0.3 3 1,0 24 6.25 ± 0.15 0.1 26 7.5 ± 0.25 0.01 29th 6.5 ± 0.15 4 1,0 23 6.5 ± 0.15 0.1 24 7.5 ± 0.3 0.01 thirty 6.5 ± 0.2

Table 6 The results of a study of the virus-neutralizing activity of the blood serum of rabbits immunized with different strains of the THES virus Strains Number of rabbits The average titers of antibodies to the THES virus (log ₂ ) after days 0 days (before immunization) 14 days 30 days "Krasnodonsky" 7 <2.0 3.75 ± 0.24 5.25 ± 0.25 "Purdue" 7 <2.0 5.83 ± 0.29 6.66 ± 0.31 Table 7 The results of a study of the immunogenic activity of an inactivated emulsion vaccine against HHPP from the strain Krasnodonsky-VNIIZZH-DEP Vaccine Antibody titers, log ₂ Sows Piglets Blood Colostrum Domolosive 2 day From the strain "Krasnodonsky-ARRIAH-DEPT" n = 5
8.75 ± 0.25 n = 5
10.2 ± 0.3 n = 5
<2.0 n = 6
8.25 ± 0.5

Claims (1)

The strain of the virus "Krasnodonsky" transmissible gastroenteritis of pigs, family Coronaviridae, genus Coronavirus, a collection of VGNKI, "Krasnodonsky - ARRIAH-DEPT", for the manufacture of diagnostic and vaccine preparations.