RU2845498C1 - Bovine viral diarrhoea virus strain of the second genotype for the manufacture of biological preparations for the diagnosis and specific prevention of bovine viral diarrhoea virus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology and veterinary virology. Strain "Chelyabinsk" of viral diarrhoea virus of cattle of second genotype of family Flaviviridae, genus Pestivirus, species Pestivirus tauri, deposited in the All-Russian State Collection of Exotic types of foot-and-mouth disease virus and other animal pathogens of the FGBI “ARRIAH” under registration number: No. 754-dep/24 GKSHM FGBU “VNIIZZH”, for the manufacture of biological preparations for the diagnosis and specific prevention of bovine viral diarrhoea.

EFFECT: invention widens the range of viral diarrhoea virus strains having infectious, antigenic and immunogenic activity.

1 cl, 9 dwg, 4 tbl, 5 ex

Description

The invention relates to the field of veterinary virology and biotechnology, in particular, to a new strain of the bovine viral diarrhea (VD) virus, which can be used in the development and manufacture of diagnostic and specific prophylactic means for VD of the second genotype of bovine viral diarrhea.

The viral diarrhea virus causes a variety of clinical forms of infection, leading to significant economic losses in beef and dairy farming worldwide. The infection is accompanied by reproductive pathology, respiratory tract diseases, immune system dysfunction, erosive and ulcerative inflammation of the mucous membranes of the digestive tract, chronic diseases with a predisposition to the development of secondary bacterial and other viral infections. Also, a distinctive feature of the virus is its ability to overcome the placental barrier and, depending on the gestational age of the cow, infect the fetus, which leads to the development of persistent infection [1]. As a result, immunotolerant calves are born, which are a constant source of pathogen for non-immune animals.

The main feature of the BVD virus is its genotypic and phenotypic diversity, which underlies the polymorphism of the clinical manifestation of the disease and changes in the virulence and antigenicity of the virus.

To date, three genotypes of the virus are known that cause this disease: VD1, VD2, VD3. At the same time, genotypes are divided into subtypes: 21 subtypes of the first genotype of VD (1a-1u), six subtypes of the second genotype (2a-2f) and four of the third are known [2].

The distribution of virus species and subtypes has regional characteristics and depends on the nature of livestock farming, stocking density, productivity, frequency of introduction of new animals and other factors.

The first genotype of the virus is widespread, but foci most often occur in European countries. Strains of the second genotype of the BVD virus are less common than strains of the first genotype of the BVD virus; they include strains that cause acute and hyperacute disease with high mortality, thrombocytopenia, and hemorrhages [3].

Cases of clinical lesions of animals caused by the bovine viral diarrhea pathogen of the second genotype have been recorded in the territory of the Russian Federation for fifteen years. Subtype 2c was detected in the Tyumen Region, subtype 2b – in the Novosibirsk Region. The detected subtypes display the same clinical picture – reproductive dysfunction (aborted fetus, stillbirth) and fetal infection of varying severity [4, 5, 6].

For the Russian Federation, there is a threat of introduction of the bovine viral diarrhea virus of the second genotype, as well as atypical viruses with susceptible animals imported from unfavorable countries, as well as products of biological origin, which requires laboratory surveillance among imported livestock [7, 8, 9].

Specific immunity plays an important role in the fight against viral diarrhea. Elimination of infection without vaccination is difficult or impossible due to widespread infection, long-term latent carriage and excretion of the virus; high population size and density of livestock on the farm. Vaccination should protect against viremia and the spread of the virus, prevent infection of target cells of the reproductive and lymphatic systems, in order to prevent fetal infection and the development of immunosuppression. Several decades ago, most vaccines contained strains of the first genotype of the virus. However, due to the antigenic variability of the virus, the introduction and production of both attenuated and inactivated vaccines based on strains of two genotypes of the virus began [10, 11].

Abroad, mainly in the USA, the strain "890" of the BVD virus of the second genotype is used in vaccines against BVD. This strain was initially isolated in 1992 from the intestine of a one-year-old heifer that died from the disease with signs of gastrointestinal damage. The results of the autopsy revealed hemorrhagic inflammation of the intestinal mucosa [12]. In 1995, a phylogenetic analysis of the strain "890" was carried out together with isolates of BVD of genotypes 1 and 2, isolated from 1960 to 1993 in the USA and Canada [13]. In the same work, the complete sequence of the strain "890" was obtained and published in the GenBank database.

Numerous strains of the second genotype have been isolated in North America. Strains 53637-2a, 296-2a, 125-2a, and 5912-2a are widely used as components of live (MLV) and inactivated vaccines by foreign companies (Hipra, Zoetis) [14].

In China, the pathogen of the second genotype was first recorded in 2004. The XJ-04 strain is highly virulent with cytopathic manifestations in cell culture. The isolate was originally isolated from a pig [15].

The Hokudai-Lab/09 strain was isolated in Japan in 2011. Strain SD1301 was identical to the Japanese Hokudai-Lab/09 strain and was assigned to group 2b [16].

In the territory of the Russian Federation, an isolate of the bovine viral diarrhea virus belonging to the second genotype was first isolated in the territory of the Novosibirsk region from an aborted cow of local origin in 2008 (isolate Blagodatsky) [17].

To date, there are no officially declared strains belonging to the second genotype on the territory of the Russian Federation.

The problem that the present invention is aimed at solving is to expand the arsenal of strains of bovine viral diarrhea virus that have high infectious, antigenic and immunogenic activity in their native form and retain antigenic and immunogenic activity after inactivation, thereby ensuring the production of sensitive and highly specific diagnostics and vaccine preparations.

The stated problem was solved by obtaining a new strain “Chelyabinsk” of the bovine viral diarrhea virus of the second genotype, which can be used for the production of diagnostic tools and specific prevention of bovine viral diarrhea.

The isolate that served as the source for obtaining the Chelyabinsk strain of the second genotype of the BVD virus was isolated in 2021 from nasal discharge samples obtained from a calf.

To obtain the Chelyabinsk strain of the bovine viral diarrhea virus of the second genotype, which has optimal biotechnological properties, a subculture of lamb testicle cells (LT) was used (Fig. 1-2). As a result of a series of successive passages on this cell culture, the Chelyabinsk strain of the bovine viral diarrhea virus of the second genotype was obtained from the previously isolated isolate. It has stable biotechnological properties and is suitable for use in laboratory studies and the manufacture of diagnostic drugs against this infection. The strain is also adapted to the continuous culture of calf kidney cells (MDBC) (Fig. 3-4) and subcultures of lamb testicle (LT), lamb kidney (LK) (Fig. 5-6) and kid kidney (KK) cells (Fig. 7-8), which makes it possible to obtain on an industrial scale the viral material of the Chelyabinsk strain of the second genotype of the bovine viral disease virus with a cytopathic activity of at least 5.0 lg TCID ₅₀ /cm ³ .

The Chelyabinsk strain of the second genotype of the BVD virus is deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Virus and Other Animal Pathogens (GKShM) of the All-Russian Research Institute of Animal Health under the registration number: No. 754 – dep / 24 – GKShM of the All-Russian Research Institute of Animal Health.

The essence of the invention is explained in graphic images:

Fig. 1. – Uninfected monolayer of TN cells;

Fig. 2. – CPE of the Chelyabinsk strain of the bovine viral load virus of the second genotype in a monolayer of TY cells (72 hours of cultivation);

Fig. 3. – Uninfected monolayer of МDBK cells;

Fig. 4. – CPE of the Chelyabinsk strain of the second genotype of the bovine viral load virus in a monolayer of MDBK cells (72 hours of cultivation);

Fig. 5. - Uninfected monolayer of NP cells;

Fig. 6. - CPE of the Chelyabinsk strain of the second genotype of the bovine viral disease virus in a monolayer of PY cells (72 hours of cultivation)

Fig. 7 - Uninfected monolayer of PC cells;

Fig. 8 - CPE of the Chelyabinsk strain of the bovine viral load virus of the second genotype in a monolayer of PC cells (72 hours of cultivation);

Fig. 9. – Phylogenetic dendrogram constructed based on the results of sequencing based on the 5’ untranslated region (5’UTR) of the bovine diarrhea virus genome.

The essence of the invention is explained by the following lists of sequences:

SEQ ID NO: 1 represents the nucleotide sequence of the 5’-UTR genome region of the Chelyabinsk strain of the bovine viral load virus.

The Chelyabinsk strain of the second genotype of the BVD virus is characterized by the following features and properties:

Morphological features

The Chelyabinsk strain of the bovine viral diarrhea virus belongs to the Flaviviridae family, Pestivirus genus, Pestivirus tauri species and has morphological features typical of the causative agent of bovine viral diarrhea: a spherical virion with a diameter of 40-60 nm consists of a nucleocapsid of icosahedral symmetry containing positive single-stranded RNA. The buoyant density of virions in a sucrose gradient is in the range of 1.12÷1.13 g/ ^cm3 , the sedimentation coefficient of virion RNA is 138S.

Antigenic properties

After the introduction of an antigen into animals, specific antibodies are formed in the blood serum, which are detected in an enzyme-linked immunosorbent assay.

Resistance to external factors

The Chelyabinsk strain of the bovine viral diarrhea virus of the second genotype is most stable at pH 7.4. It is quickly inactivated at pH 3. When the pH of the medium changes from 5.7 to 9.3, infectivity decreases.

The virus is well preserved in its native form at 4°C, minus 20°C and minus 40°C. The virus is sensitive to ether, chloroform, trypsin, acidic pH (3.0) and sodium deoxycholic acid; at 37°C it dies after 5 days.

Lyophilization has almost no effect on its activity, but repeated freezing and thawing reduces the virulence and immunogenicity of the virus.

Genetic and taxonomic characteristics

The Chelyabinsk strain of the bovine viral diarrhea virus is represented by a positive single-stranded RNA of about 12,500 nucleotides in size with a single open reading frame. The open reading frame begins with nucleotide 386. All information about proteins is contained in one reading frame.

The RT-PCR method was used to amplified a region of the bovine diarrhea virus genome, including 217 nt of the 5'-UTR region. Phylogenetic characteristics established that the genetic fragment of the virus belongs to the second genotype (Fig. 1) [18].

Biotechnological characteristics

The Chelyabinsk strain of the second genotype of the bovine viral disease virus reproduces in a continuous culture of calf kidney cells (MDBK), in subcultures of lamb testicle (LT), lamb kidney (LK) and kid kidney (KK) cells. Reproduction of the virus is accompanied by a specific cytopathic effect, leading to cell rounding and the subsequent formation of granular foci of affected cells on the surface of the monolayer.

At 37°C during 72÷96 hours of incubation, the virus accumulates in titers of 4.5÷5.0 lg TCID ₅₀ /cm ³ and retains its original characteristics when passaged in sensitive biological systems for at least 5 passages (observation period).

Additional features and properties

Antigenic activity - has antigenicity in the composition of preparations for hyperimmunization of donors.

Reactogenicity - does not have reactogenic properties.

Stability - retains the original biological (antigenic) properties when passaged in the culture of MDBK, PY, PK, TY cells for 5 serial passages.

Pathogenicity - not pathogenic for guinea pigs, rabbits and white mice.

Virulence is expressed.

Free from contamination by bacteria, mycoplasma and foreign viruses.

The essence of the proposed invention is explained by examples of its use, which do not limit the scope of the invention.

Example 1. Biological and virological studies of the strain.

Biological and virological methods included adaptation of the virus to cell cultures of TYa, MDBC, PYa and PC.

To obtain the Chelyabinsk strain of BVD virus, the viral suspension was added to the cell culture monolayer freed from the growth medium and exposed for one hour at a temperature of (37.0±0.5)°C. After that, the semi-synthetic maintenance medium (SSM) was added. The infected culture was incubated at a temperature of (37.0±0.5)°C until the cytopathic effect (CPE) of the virus appeared, which was characterized by rounding of the cells, their accumulation in the form of aggregates of different sizes, total destruction, and detachment of the cell monolayer from the substrate did not occur (Figs. 1-8). When at least 60% of the cell monolayer area was affected, the culture flasks were frozen at minus (80.0±4.0)°C and after thawing at room temperature, the virus was collected, followed by sampling to exclude microbial contamination and determine the infectious activity of the virus by microtitering in the TY cell culture.

Titration was carried out in sterile 96-well flat-bottomed culture plates in a volume of 0.1 ^cm3 /well. Beforehand, 0.2 cm3 of cell suspension prepared in the PSP nutrient medium with a cell concentration of
0.3-0.4×10 ⁶ cells/cm³, after which the plate was incubated at a temperature of (37.0±0.5)°C with 5% CO ₂ for 24 h, with daily monitoring of the monolayer confluence: the wells of the plate had to contain a fully formed cell monolayer with no signs of degeneration. Next, ten-fold dilutions of the virus-containing material were prepared on the PSP medium, starting from 10 ^-1 to 10 ^-8 . The prepared dilutions of the virus were transferred, starting with the highest dilution, by 0.1 cm³ into the wells of the culture plate previously washed from the growth medium. The plate was incubated in a CO ₂ incubator containing 5% CO ₂ at a temperature of (37.0±0.5)°C. The final accounting of the virus titration results was carried out after 120-144 hours of incubation, provided that the integrity of the cell monolayer in the control wells was maintained. The calculation of infectious activity was carried out using the Reed and Mench method and expressed in lg TCD ₅₀ /cm ³ .

The results of virus adaptation to various cell cultures are presented in Table 1. The infectious activity of the virus varied from 5.05±0.13 to 5.3±0.14 lg TCID ₅₀ /cm ³ .

Example 2. Species affiliation of the Chelyabinsk strain of the second genotype of the bovine viral disease virus.

To determine the species affiliation of the Chelyabinsk strain of the bovine viral disease virus, the following oligonucleotide primers were used: BD1 for the NADL strain - positions 188-205 nt, BD4 for the NADL strain - positions 383-366 nt, BD2 for the 890 strain - positions 204-223 nt, BD3 for the 890 strain - positions 320-301 nt, flanking the conservative region of the viral genome measuring 261 base pairs, and a method for detecting and differentiating the pathogen by strains in the 5’-UTR region of the genome in a Rotor-Gene 6000 programmable amplifier [19].

The conducted phylogenetic analysis of the virus showed that the “Chelyabinsky” strain belongs to the second genotype and has 98% homology with the reference strain “890” of subtype 2a of the BVD virus (Fig. 9).

Example 3. Control of the stability of the biological activity of the strain

The stability control of the biological activity of the strain was determined during 5 consecutive passages in the cultures of TYa and MDBK cells. The results of the study of the stability of the strain by its biological activity during 5 passages are presented in Table 2.

In the course of the conducted studies it was established that the Chelyabinsk strain of the second genotype of the bovine viral disease virus demonstrated stable biological activity during 5 consecutive passages in TYa and MDBK cell cultures, which was within the range of 5.0±0.19 - 5.56±0.08 lg TDC ₅₀ /cm ³ .

Example 4. Obtaining an antigen of the Chelyabinsk strain of the bovine viral disease virus.

To prepare the antigen, the Chelyabinsk strain of the BVD virus was used to inoculate a two-day culture of TY cells grown in 1.5-liter Wedge mattresses, after draining the growth medium, with a virus-containing suspension at a dose of at least 0.01 TCID ₅₀ /cell. The mattresses were placed in a thermostat at a temperature of (37 ± 0.5) °C for one hour to bring the culture cells into contact with the virus. After that, the PSP maintenance medium was added. The infected cell culture was incubated at a temperature of (37.0 ± 0.5) °C until the CPE of the virus appeared. When at least 60% of the monolayer area was affected, the culture mattresses were frozen three times at a temperature of minus (80.0 ± 1.0) °C and thawed.

The resulting virus-containing culture fluid was purified from ballast proteins and cell fragments by low-speed centrifugation at 4800 g for 40 minutes.

The supernatant was collected (eluate 1), and the pellet was resuspended in TNE buffer to a final suspension volume equal to 1% of the volume of the original virus-containing material. To release the remaining virus from the cells, the pellet was frozen and thawed twice, after which it was centrifuged under the same conditions to obtain the 2nd, 3rd, and 4th eluates.

The resulting supernatants (eluates 1,2,3,4) were then combined and centrifuged at 6000 g for 30 min, the supernatant was removed, and the pellet was resuspended in 1/100 of the original volume of TNE buffer. 20% sucrose was added under the resulting suspension and centrifuged at 10000 g for 2 h. The supernatant was poured off, and the pellet was resuspended in TNE buffer.

The data on the assessment of the material activity at each stage of purification and concentration are shown in Table 3.

As a result, a purified preparation of the antigen of the Chelyabinsk strain of the bovine viral infection virus with an infectious activity of 7.1 lg TCID ₅₀ /cm ³ was obtained.

The antigen of the Chelyabinsk strain of the bovine viral disease virus obtained by this method was used as a biopreparation to obtain hyperimmune virus-specific blood serums of rabbits containing antibodies against the Chelyabinsk strain of the bovine viral disease virus.

Example 5. Obtaining virus-specific blood serums against the Chelyabinsk strain of bovine viral diarrhea virus

To obtain specific blood serums against the Chelyabinsk strain of bovine viral diarrhea virus, 5 rabbits weighing 2.0-2.5 kg were used. For hyperimmunization of animals, the Chelyabinsk strain antigen of the bovine viral diarrhea virus was used, obtained as described in Example 3, from which a complex emulsion was prepared using the oil adjuvant Montanide ISA-70 (Seppic, R. France) (in the ratio of adjuvant/antigen = 70/30 by weight).

The obtained vaccine preparation was used to immunize rabbits at a dose of 2 ^cm3 three times according to the following scheme:

1) 1 immunization - into the soft pads of the hind paws;

2) 10 days after the first immunization into the popliteal lymph nodes;

3) 21 days after the first – intramuscularly.

Before each immunization, blood was collected from the ear vein. The tubes with blood were placed in a thermostat at 37°C until a blood clot formed. The blood clot was separated and placed overnight at +4°C. The following day, the supernatant fraction, blood serum, was collected and centrifuged at 2,000 rpm for 30 minutes. The resulting blood serum was used in ELISA to determine the level of antibodies against the Chelyabinsk strain of BVD.

Ten to fifteen days after the end of immunization, donors were bled and their blood serum was obtained for testing for antibodies in ELISA. The results of the ELISA testing of blood serum are presented in Table 4.

Analyzing the ELISA results in Table 4, we can conclude that on the 35th day after triple immunization, the production of specific antibodies is observed in animals; the titer of specific antibodies is 1:6400-1:12800.

The conducted studies have shown the possibility of using the Chelyabinsk strain of the bovine viral disease virus as a biopreparation for obtaining diagnostic serums with antibody activity of at least 1:6400-1:12800.

The volume of specific antibodies against the bovine viral diarrhea virus of the second genotype obtained during the studies shows that biopreparations manufactured using the Chelyabinsk strain of the bovine viral diarrhea virus can potentially be used to create specific immunity against bovine viral diarrhea in naturally susceptible animals.

Thus, the Chelyabinsk strain of the bovine viral diarrhea virus of the second genotype obtained in accordance with the proposed invention has high biological and antigenic activity, is suitable for the manufacture of biopreparations for diagnostics and specific prevention, and expands the arsenal of new domestic production strains of the bovine viral diarrhea virus.

Sources of information taken into account when drafting the description of the invention for the application for the issuance of a patent of the Russian Federation for the invention "Chelyabinsk strain" of the bovine viral diarrhea virus of the second genotype for the manufacture of biopreparations for the diagnosis and specific prevention of bovine viral diarrhea virus"

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16.

Table 1

Biological properties of the Chelyabinsk strain of the second genotype of the bovine viral disease virus

n=3

System
reproductions
virus Time
manifestations
biological
activities
(watch) Quantity
adaptation
passages Characteristic
adapted
material Virus titer, lgTCD ₅₀ /cm ³ TY 72 5 5.3±0.14 MDBK 96 2 5.25±0.11 ПЯ 72 2 5.05±0.13 PC 72 2 5.15±0.16

Table 2

Stability of the Chelyabinsk strain of the second genotype of bovine viral disease virus during reproduction in cell cultures

n=3

Passage No. Virus titer, lg TCD ₅₀ /cm ³ Cell culture TY MDBK 1 5.1±0.16 5.0±0.19 2 5.3±0.14 5.25±0.14 3 5.25±0.11 5.35±0.11 4 5.34±0.17 5.48±0.12 5 5.37±0.15 5.56±0.08

Table 3

Evaluation of the activity of the BVD viral material at different stages of purification

Sample No. Characteristics of the material under study Infectious activity titer
(lg TCD ₅₀ /cm ³ ) PCR 1 original, culture virus of bovine viral disease (4th passage in CC TY) 5.0 + 2 cellular detritus 4.0 + 3 combined supernatants (eluates) 3.5 + 4 concentrate of eluates after 30% sucrose 7.1 +

Claims (1)

Strain "Chelyabinsk" of the bovine viral diarrhea virus of the second genotype familiesFlaviviridae, kindPesticides, typePestivirus tauri, deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Virus and Other Animal Pathogens of the Federal State Budgetary Institution “All-Russian Research Institute of Animal Health” under registration number: No. 754 – dep / 24 GKShM of the Federal State Budgetary Institution “All-Russian Research Institute of Animal Health”, for the manufacture of biopreparations for the diagnosis and specific prevention of viral diarrhea in cattle.