RU2799604C1 - Lumpy skin disease virus of the capripoxvirus genus neethling-arriah strain for the manufacture of biological products for the specific prevention of infectious nodular dermatitis in cattle - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a strain of the virus of infectious nodular dermatitis in cattle. The described Neethling-ARRIAH strain of infectious nodular dermatitis virus was isolated in 2020 from samples of biological material taken on the territory of the Republic of Bashkortostan from cattle suspected of being infected with infectious nodular dermatitis of cattle, and deposited in the All-Russian State Collection of Exotic Types of FMD Viruses and Other Animal Pathogens (GKShM) FGBI "ARRIAH" under registration number: No. 393 - dep./22-27 — GKSHM FGBI "ARRIAH". The strain reproduces in TU, TSN and PB cell cultures for 3–5 days and accumulates in a titer of at least 10^4.5 TCD₅₀/cm³, retains its original characteristics when passaged in TU, TSN and PB cell cultures for 5 passages.

EFFECT: obtaining Lumpy skin disease virus of the Capripoxvirus genus Neethling-ARRIAH strain for the manufacture of biological products for the specific prevention of infectious nodular dermatitis in cattle.

1 cl, 13 dwg, 3 tbl, 6 ex

Description

The invention relates to the field of veterinary virology and biotechnology, in particular, to a new strain of infectious nodular dermatitis virus, which can be used for the manufacture of biological products for the specific prevention of infectious nodular dermatitis in cattle.

Infectious nodular bovine skin dermatitis virus (bovine LSD virus) belongs to the Poxviridae family of the Capripoxvirus genus and is the causative agent of a dangerous transboundary disease that poses a serious threat to cattle breeding worldwide [1, 2, 3, 4]. Causing huge economic losses due to trade restrictions and culling of sick animals, as well as potentially dangerous livestock products, LSD in cattle is subject to mandatory notification to the OIE [5, 6].

Cattle of all ages (Bos taurus, Bos indicus) and Asiatic buffaloes (Bos bubalis) are susceptible to the bovine itch virus. When the infection circulates in the herd, there is a decrease in milk yield, in pregnant cows, abortions and problems during subsequent insemination are possible. The disease of animals is accompanied by fever and the appearance of skin lesions growing deep into the underlying tissues, including muscle tissue [7, 8, 4].

Skin lesions are necrotic areas with fibrin deposits and contain high concentrations of the virus, being a source of environmental contamination with viral material. The mechanisms of transmission of the causative agent of LSD in cattle are currently poorly understood. It is tentatively believed that the virus is transmitted through the bites of blood-sucking insects, while other transmission mechanisms, such as contact or aerial transmission, are considered to be ineffective due to the lack of supporting experimental data [9, 10, 11].

The wide spread of the bovine LSD virus in new territories of the Northern Hemisphere emphasizes the objective need to study both the biological properties of the pathogen in new climatic conditions, including possible ways of infection and spread, and the genetic and biological properties of the virus in order to develop an effective strategy and control, diagnosis, prevention and eradication of infection.

In the Russian Federation, infectious nodular dermatitis was first registered in July 2015, when 17 foci of infectious nodular bovine dermatitis were detected in three regions of the North Caucasus Federal District, incl. 11 - in the Republic of Dagestan, 4 - in the Chechen Republic, 2 - in the Republic of North Ossetia.

In 2016, the disease reached an unprecedented level of prevalence, with 313 outbreaks notified to the OIE. Not only the number of outbreaks has increased tremendously (compared to the previous year), but also the number of disadvantaged regions. Thus, trouble was recorded in seventeen regions of the Southern, North Caucasian, Central and Volga Federal Districts.

In 2017, 43 foci of lumpy dermatitis of cattle were recorded in 6 regions of the Russian Federation: Saratov region - 24, Orenburg region - 11, Volgograd region - 3, Samara region - 3, Republic of Bashkortostan - 1 and Ulyanovsk region - 1.

In 2018, 64 foci of lumpy dermatitis of cattle were recorded in 6 regions of the Russian Federation: Samara region - 32, Kurgan region - 21, Chelyabinsk region - 4, Omsk region - 5, Sverdlovsk region - 1 and Saratov region - 1.

In 2019, 29 foci of lumpy skin disease of cattle were recorded, most outbreaks are recorded along the border with the Republic of Kazakhstan (Udmurt Republic, Saratov Region, Omsk Region, Novosibirsk Region, Tyumen Region and Altai Territory).

In 2020, the distribution of LSD in cattle was detected in an easterly direction along the Chinese border. Thus, outbreaks were detected in the Tomsk region - 1 focus, Altai Territory - 1 focus, Khabarovsk Territory - 4 centers and the Jewish Autonomous Region - 1 focus. Genetic analysis of the isolates identified in 2020 showed that they all belong to the genetic line of the Neethling type of the group of vaccine-like isolates. It is important to note that, starting from 2017, bovine LSD vaccine-like virus tends to spread along the border of the Russian Federation with the Republic of Kazakhstan, where a homologous vaccine is used. Interestingly, in 2019, vaccine-like isolates of the bovine LSD virus were detected in China, 20 km from the border in the Republic of Kazakhstan and 700 km from the border in the Russian Federation. In this regard, there are high risks of further spread of the bovine LSD virus in the region, including the Far East of the Russian Federation.

In 2021, on the territory of the Russian Federation, outbreaks of the disease were diagnosed on the territory of the Trans-Baikal Territory - 34 outbreaks, which borders Mongolia and China, as well as on the territory of the Republic of Bashkortostan - 4 outbreaks, the Chelyabinsk region - 1 outbreak and the Republic of Buryatia - 3 outbreaks.

In 2022, outbreaks were recorded in the Republic of Buryatia - 9 outbreaks, the Republic of Tatarstan - 1 outbreak, the Amur Region - 1 outbreak and the Republic of Tyva - 2 outbreaks.

LSD in cattle is classified as a particularly dangerous animal disease that can cause epizootics and cause significant economic damage to livestock [12]. In accordance with the new classification of the International Epizootic Bureau (OIE), the disease is included in the list of diseases subject to mandatory notification (category “Diseases and infections of cattle”) [13, 14, 19].

The causative agent of infection is a DNA-containing enveloped virus that is antigenically related to the sheep pox and goat pox viruses, but differs from them at the genetic level, and together with them forms an independent genus Capripoxvirus of the Poxviridae family [13, 15, 17].

Currently known is the strain "VND bovine/Dagestan/2015" of the bovine LSD virus isolated in 2015 from samples taken from infected cattle in the territory of the Republic of Dagestan. This strain belongs to the cluster of field isolates. It is reproduced in a continuous culture of domestic goat ovary cells (YADK-04) and a subculture of lamb testicles (TU), with an infectious activity of 10 ^4.5 - 10 ^5.5 TCD ₅₀ /cm ³ and is used for virological and molecular genetic studies, the manufacture of drugs diagnosis of LSD in cattle and quality control of production batches of vaccines against LSD in cattle [16]. The disadvantage of this invention is its high degree of identity with field isolates circulating in Africa, the Middle East and Europe.

Known is the Volga strain of the bovine LSD virus, isolated from pathological material from cows with clinical signs of bovine LSD, selected in the Volga Federal District of the Russian Federation in 2017; lamb testicles (TU) reproduced in the subculture, with an infectious activity of 4.5 - 5.5 lg TCD ₅₀ /cm ³ and used for virological, molecular genetic, monitoring studies, and the manufacture of diagnostic preparations [18]. This strain is a new genetic variant resulting from natural recombination between a commercial vaccine strain and the Kenyan vaccine strain "KSGP".

The Tyumen/2019 strain is known, which was obtained from samples of biological material taken in the territory of the Tyumen region of the Russian Federation from cattle with clinical signs of LSD in cattle in 2019. According to the RPO30 gene encoding the RNA polymerase subunit, this strain clusters with field isolates, while according to the gene encoding the G-protein of the chemokine receptor, the Tyumen/2019 strain of the bovine LSD virus belongs to vaccine strains [14].

All of the above strains are virulent and capable of causing clinical infection in naturally susceptible animals. The problem to be solved by the present invention is to obtain a non-virulent production strain of bovine itch virus capable of forming a protective immune response against infection with bovine itch.

This problem was solved by obtaining a new strain of "Neethling-ARRIAH" virus of infectious nodular bovine dermatitis, which can be used as the main substance in the production of specific prophylaxis of infectious nodular bovine dermatitis for naturally susceptible animals.

The bovine lumpy skin disease virus isolate, which served as a source for obtaining the Neethling-ARRIAH strain, differs from all virus strains previously isolated in Russia by the absence of virulent activity and the ability to form an immune response against bovine LSD in immunized animals. The isolate was isolated in 2020 from samples of biological material taken on the territory of the Republic of Bashkortostan from cattle suspected of being infected with LSD in cattle.

To obtain a strain of "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of cattle, which has optimal biotechnological properties, a subculture of cells of the testicles of the lamb (TU) was used. As a result of a series of successive passages on this cell culture, from the previously isolated isolate, the Neethling-ARRIAH strain of infectious nodular bovine skin dermatitis virus was obtained, which has stable biotechnological properties and is suitable for use in laboratory research and the manufacture of biological products for the specific prevention of LSD in cattle. The strain is adapted to a transplantable culture of domestic goat ovary cells (TSN) and a transplantable culture of sheep kidney cells (RB), which makes it possible to obtain on an industrial scale the viral material of the Neethling-ARRIAH strain of infectious nodular skin dermatitis virus with a cytopathic activity of at least 10 ^5.0 TCD ₅₀ / cm ³ .

The strain of infectious nodular bovine dermatitis virus "Neethling-ARRIAH" was deposited in 2022 in the All-Russian State Collection of Exotic Types of Foot and Mouth Virus and Other Animal Pathogens (GKSHM) FGBI "ARRIAH" - under registration number: No. 393 - dep / 22-27 - GKShM FGBU "ARRIAH".

The essence of the invention is illustrated in the graphic images:

Fig. 1. - Uninfected monolayer of TU cells;

Fig. 2. - CPD of the strain "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of cattle in a monolayer of TU cells (48 hours of cultivation);

Fig. 3. - CPD of the Neethling-ARRIAH strain of infectious nodular bovine dermatitis virus in a monolayer of TU cells (96 hours of cultivation);

Fig. 4. - TU cells affected by the Neethling-ARRIAH virus strain;

Fig. 5. - Uninfected monolayer of TSN cells;

Fig. 6. - CPD of the strain "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of cattle in a monolayer of TCH cells (48 hours of cultivation).

Fig. 7. - CPD of the strain "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of cattle in a monolayer of TCH cells (96 hours of cultivation);

Fig. 8. - Indicators of thermometry of cattle in the study of the virulent and immunogenic properties of the strain "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of cattle;

Fig. 9 - Animals prepared for titration of the Privolzhsky strain of the bovine LSD virus;

Fig. 10 - The process of titration of the strain "Privolzhsky" of the LSD virus;

Fig. 11 - An example of the absence of a tissue reaction to the introduction of the Privolzhsky strain of the bovine itch virus in an individual immunized with the Neethling-ARRIAH strain of the bovine itch virus (7 days after the start of titration);

Fig.12 - An example of a tissue reaction to the introduction of the strain "Privolzhsky" virus LSD in cattle not immunized with the strain "Neethling-ARRIAH" virus LSD in cattle individuals (7 days after the start of titration);

Fig. 13 - Dendrogram reflecting the phylogenetic relationship of the Neethling-ARRIAH strain (LSDV Bashkortostan Neethling) based on genome-wide sequences of capripoxviruses.

The Neethling-ARRTAH strain of infectious nodular bovine dermatitis virus is characterized by the following features and properties:

Morphological features

The "Neethling-ARRIAH" strain of bovine skin disease virus belongs to the class Pokkesviricetes, order Chitovirales, family Poxviridae, subfamily Chordopoxivirinae, genus Capripoxvirus, species Lumpy skin disease virus. The strain has morphological features typical for representatives of capripoxviruses: virions are brick-shaped, have a double lipid membrane, a dense core and lateral bodies.

Antigenic properties

The introduction of the strain "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of cattle is not correlated with the formation of virus-neutralizing antibodies in the blood serum of naturally susceptible animals.

In this case, the virus is stably neutralized by cattle serum specific to infectious nodular dermatitis.

Pathogenic properties

The strain is not pathogenic for animals naturally susceptible to infectious nodular dermatitis of cattle. During experimental infection of 1.5-year-old bulls with a virus-containing suspension for 21 days, the development of clinical signs characteristic of infectious nodular dermatitis of cattle was not observed.

Immunogenic properties

The introduction of naturally susceptible animals of the strain "Neethling-ARRIAH" of the virus of infectious nodular bovine dermatitis causes them to form a protective immune response against infection with virulent forms of the bovine LSD virus.

Contamination with bacteria, fungi, mycoplasmas and foreign viruses

The strain is not contaminated with bacteria, fungi, mycoplasmas and foreign viruses.

Biotechnological characteristics

The Neethling-ARRIAH strain of bovine irritant skin disease virus propagates in a subculture of lamb testicle (TU) cells, in a continuous culture of domestic goat ovary cells (YADK-04), and a continuous culture of ram kidney cells (RB) [19]. The reproduction of the virus is accompanied by a specific cytopathic effect, leading to rounding, cell aggregation and destruction of the monolayer on the 2nd - 3rd day of cultivation. When cultivated, the Neethling-ARRIAH strain accumulates in a titer of at least 10 ^4.5 TCD ₅₀ /cm ³ and retains its original characteristics when passaged in sensitive biological systems for at least 5 passages (observation period).

Molecular genetic characterization

According to the analysis of the GPCR gene encoding the G protein of the chemokine receptor, the Neethling-ARRIAH strain of bovine irritant skin disease virus belongs to vaccine strains.

Whole genome analysis showed a closer nucleotide homology of the strain "Neethling-ARRIAH" of the bovine LSD virus with vaccine strains by 99.9% (Fig. 13).

Physical properties

The floating density in CsCl is 1.19 g/cm ³ . Most stable at pH 6.6 - 8.6.

Resistance to external factors

The virus is stable in the environment. At room temperature, it retains its infectious activity for at least 18 days, in the affected areas of the skin - at least 33 days, in saliva - 11 days, in bull sperm - 22 days. In culture liquids, the strain is stable for 6 months at 4°C. Tolerates 3 times freezing and thawing. Sensitive to ether, chloroform and high temperature. At a temperature of minus 80°C it remains viable for 10 years.

Additional features and properties

Stability - retains the original biological properties when passaged in cell cultures of TU, TSN and PB for at least 5 consecutive passages (observation period).

Pathogenicity - Not pathogenic to naturally susceptible animals.

Virulence - not virulent in naturally susceptible animals.

Immunogenicity - causes the formation of an immune response against clinical infection with the virus of infectious nodular dermatitis of cattle.

Free from contamination by bacteria, mycoplasmas and foreign viruses.

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

Example 1 Biological and Virological Studies of the Strain

Cell lines sensitive to virulent strains of bovine bovine skin disease virus were used to determine cell cultures sensitive to the Neethling-ARRIAH strain: lamb testicle (TU) cell subculture; transplantable cell culture of domestic goat testicles (TSN); transplantable culture of sheep kidney cells (RB).

The virus-containing suspension was applied to a cell culture monolayer freed from the growth medium and exposed for an hour at a temperature of (37.0±0.5)°C. After that, a PSP maintenance medium was added with the addition of 1–2% of fetal cattle blood serum. The infected culture was incubated at a temperature of (37.0±0.5)°C until the cytopathic effect (CPE) of the virus appeared. To control the cell culture, two uncontaminated vessels were left: one with the initial growth medium, the other with the supporting medium.

Control vessels and vessels with an infected culture were incubated at a temperature of (37.0±1.0)°C until the appearance of CPE of the virus in more than 80% of the cells of the monolayer (72-96 hours).

To extract the virus from the cells during their destruction, the contents of the vessels were frozen once at a temperature of minus (20.0±1.0)°C for 24 h, after which they were thawed. Four successive passages were carried out by the described method.

Determination of the degree of accumulation of the cytopathic activity of the strain "Neethling-ARRIAH" of the infectious nodular dermatitis virus in bovine animals in the obtained successive passages was carried out by microtitering in 96-well culture plates in the subculture of TU cells.

To do this, a series of tenfold dilutions in PSP medium (from 10 ^-1 to 10 ^-7 ) was prepared from a sample of the combined virus-containing material of each passage.

The prepared dilutions (starting from the highest) were transferred into culture plates (introduced dilution volume - 0.1 cm ³ ), 4 wells for each dilution. Then, 0.1 cm ³ of a cell suspension of the TU subculture (cell concentration 0.4-0.5×10 ⁶ cells/cm ³ ) prepared in PSP medium containing 1-2% fetal blood serum was added to the virus dilutions distributed in the indicated way. KRS.

Microscopy infected with a strain of "Neethling-ARRIAH" virus of infectious nodular dermatitis of cattle cell culture showed that the CPD of this strain is characterized by rounding of cells, their accumulation in the form of aggregates of different sizes with further total destruction and exfoliation of the monolayer of cells from the substrate (figure 1, figure 2 , figure 3, figure 4, figure 5, figure 6, figure 7).

At the same time, it should be noted that the CPE of the studied isolate began to manifest itself most clearly at the level of passages 3 and 4, which indicates the adaptation of the virus to cell culture production lines.

To control the quality of TU subculture cells, at least 4 wells of a culture plate were used, into which 0.1 cm ³ of the PSP medium and 0.1 cm ^{3 of} the TU subculture cell suspension were added (cell concentration 0.4-0.5 × 10 ⁶ cells/ cm ³ ) prepared in PSP medium containing 1-2% fetal bovine serum.

Prepared tablets were incubated at a temperature of (37.0±0.5)°C in the presence of 5% CO ₂ in an atmosphere. The results of titration were taken into account after 120-168 hours of incubation using an inverted microscope, marking wells with signs of manifestation of CPE of the infectious nodular dermatitis virus of cattle. The calculation of the cytopathic activity of the strain "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of cattle was carried out according to the method of Reed and Mench and expressed in TCD ₅₀ /cm ³ (table 1).

The results of the titration showed that by the fifth passage in all selected cell lines the cytopathic activity of the strain "Neethling-ARRIAH" of the infectious nodular skin dermatitis virus of cattle is at a level of at least 10 ^5.00 TCD ₅₀ /cm ³ , which proves the sensitivity of these cell cultures to the strain "Neethling-ARRIAH" virus of infectious nodular bovine dermatitis.

Example 2 Stability Study of a Strain

The stability of the phenotype of the Neethling-ARRIAH strain of infectious nodular bovine skin dermatitis virus was determined by five successive passages in the subculture of TU cells. The source material for titration was a virus-containing material obtained as a result of the fourth passage of the strain "Neethling-ARRIAH" of the infectious nodular dermatitis virus of bovine animals in the corresponding cell culture.

Monolayer cultures of TU, TSN, and PB cells grown with the addition of 5% fetal bovine serum in culture mats with a volume of 50 ^cm3 were used in the work. The QC used was free from viral, bacterial and other contamination, the cell monolayer was fully formed and had a characteristic pattern.

A monolayer cell culture grown with the addition of 5% fetal bovine serum was washed twice with Hank's buffer solution, pH 7.0-7.2. After that, 1.0 cm ³ of a virus-containing suspension (not less than 0.01 TCD ₅₀ /cell) was added to the culture mattresses, and placed in a thermostat at a temperature of (37.0±1.0)°C for 1 hour, for contact of the virus with cells. Then, a supporting nutrient medium (PSP or analogues) containing 2% fetal bovine serum was poured into the culture vessels. To control the cell culture, two uncontaminated vessels were left: one with the initial growth medium, the other with the supporting medium.

Vessels were incubated at a temperature of (37.0 ± 1.0)°C until the appearance of CPE of the virus in more than 80% of the cells of the monolayer (72-96 h), after which, to extract the virus from the cells during their destruction, the contents of the vessels were frozen once at temperature minus (20.0±1.0)°C for 24 hours and thawed.

Five successive passages were carried out in the manner described. The infectious activity of the virus-containing material obtained after each passage was determined by titration using the method of Reed and Mench and expressed in TCD ₅₀ /cm ³ .

The results of titration of five successive passages of the Neethling-ARRIAH strain of bovine nodular skin dermatitis virus in TU, TSN and PB cell cultures showed that during five successive passages: in the TU cell culture, the titer of the cytopathic activity of the Neethling-ARRIAH strain ranged from 10 ^5.25 to 10 ^5.43 TCD ₅₀ /cm ³ (mean titer 10 ^5.362±0.062 TCD ₅₀ /cm ³ ); in cell culture TSN - within 10 ^5.16 to 10 ^5.25 TCD ₅₀ /cm ³ (average titer 10 ^5.203±0.031 TCD ₅₀ /cm ³ ); in PB cell culture - within 10 ^5.16 to 10 ^5.17 TCD ₅₀ /cm ³ (average titer 10 ^5.198±0.029 TCD ₅₀ /cm ³ ) (Table 2).

Thus, this strain of "Neethling-ARRIAH" virus of infectious nodular dermatitis of cattle is stable when cultivated in cell cultures of TU, TSN and PB.

Example 3. Control of the absence of contamination by bacteria, fungi and mycoplasmas (microbiological methods).

For the test of sterility, 5 bottles with a sample of the initial material of the strain "Neethling-ARRIAH" of the bovine LSD virus were used. Confirmation of the absence of contamination by bacterial, fungal microflora and mycoplasmas was carried out in accordance with GOST 28085-2013 “Biological medicinal products for veterinary use. Method of bacteriological control of sterility” [21].

The contents of the vials were dissolved in sterile saline to the original volume before lyophilization (2 cm ³ ).

Suspension from each vial of 1 ^cm3 was seeded into 3 test tubes with thioglycol medium. Two inoculated test tubes were kept in a thermostat for 14 days: one - at (21±1)°С, another - at (37±0.5)°С, and the third - was kept for 7 days at (37±0.5 )°С. Then it was reseeded in 0.5 cm ³ by one test tube on the following media: MPA, MPPB, MPB, Saburo liquid and 1 cm ³ on MPPB.

Transfers to MPA, MPB, MPPB were kept for another 7 days at (37±0.5)°C, and transfer to Sabouraud - at (21±1)°C.

During the microbiological testing of the strain sample, the sterility of the media was monitored: three test tubes with each medium were kept in a thermostat for 14 days at (37±0.3)°C, with the Sabouraud medium at (21±1)°C.

To exclude contamination with mycoplasmas, a 0.5 cm ³ strain sample suspension was inoculated into a test tube containing 4.5 cm ³ of liquid Kagan medium 0.3% and kept in a thermostat at (37±0.5)°C.

In the absence of growth of mycoplasmas for 7 days on a liquid medium (preservation of the color of the indicator), then every 7 days, 2 consecutive passages were carried out on a solid medium Kagan 1.3%.

In the absence of mycoplasma-specific colonies growing into the agar thickness, a conclusion was made about the purity of the strain sample. Inoculations of the resuspended sample of the virus strain on the appropriate nutrient media were without germs during all periods of observation.

As a result of the research, it was found that in the studied initial seed material of the Neethling-ARRIAH strain of infectious nodular bovine dermatitis virus, contamination with bacteria, fungi and mycoplasmas is absent.

Example 4. Study of the virulent properties of the strain

In the experiment, 7 heads seronegative to the virus of infectious nodular bovine dermatitis were used, 5 of which (No. 1, No. 2, No. 3, No. 4 and No. 6) were injected by intravenous injection of 2 cm ³ of the virus-containing material of the Neethling-ARRIAH strain (cytopathic activity 10 ^4.67 TCD ₅₀ /cm ³ ). 2 out of 7 heads (No. 5 and No. 7) were not subjected to the injection procedure (contact animals).

After infection for 28 days, all animals in the group were clinically monitored with thermometry, noting signs of the development of the clinical picture of infectious nodular bovine dermatitis. Every 3 days, samples of stabilized blood and samples of nasal swabs were taken from all animals, followed by determination of the presence of the bovine nodular dermatitis virus genome.

28 days after the start of the experiment, by intravenous injection of 2 cm ³ of a virus-containing material of the virulent strain "Privolzhsky" of the virus of infectious nodular dermatitis of cattle (cytopathic activity of ^105.00 TCD ₅₀ /cm ³ ), all naturally susceptible animals (including control) were infected.

Within 14 days after infection, all animals in the group were clinically observed with thermometry, noting signs of the development of the clinical picture of infectious nodular bovine dermatitis. Every 3 days, samples of stabilized blood and samples of nasal swabs were taken from all animals, followed by determination of the presence of the bovine nodular dermatitis virus genome.

14 days after infection of cattle with the virulent strain "Privolzhsky" of the infectious nodular dermatitis virus, cattle were euthanized in all individuals of the group, followed by post-mortem autopsy and sampling of the prescapular lymph nodes and lymph nodes of the knee fold, which were examined by PCR-RT for the presence of the genome infectious nodular dermatitis virus of cattle.

Clinical observations for 28 days after injection of the strain "Neethling-ARRIAH" of the virus of infectious nodular bovine dermatitis showed that the animals (both injected and control) did not show clinical signs of infectious nodular bovine dermatitis. The body temperature of all individuals remained within the physiological norm.

Clinical observation for 14 days after infection of animals with a virulent strain "Privolzhsky" of infectious nodular bovine dermatitis virus revealed the development of clinical signs of infectious nodular bovine dermatitis in individuals No. 5 and No. 7 (control animals).

The first signs of the disease of infectious nodular bovine dermatitis in bull No. 5 began to appear on the fourth day after the introduction of the virulent strain in the form of single nodular formations on the skin (from 0.3 to 0.5 cm in diameter) in the region of the left and right shoulder blades. Over time, the number, diameter and area of nodular lesions continuously increased and on the sixth day after infection (35 days of experience) the bull No. 5 was completely affected by painful nodules (diameter from 3.0 to 5.5 cm). However, palpation did not reveal enlargement or tenderness of the superficial lymph nodes. Starting from the 12th day after the procedure for introducing the Privolzhsky strain of infectious nodular bovine skin dermatitis virus (8 days after the first sign of bovine skin disease), the nodules began to decrease in size and became painless by 14 days after infection. The body temperature of the animal exceeded the physiological norm for almost the entire period of observation after infection with a virulent strain of the LSD virus, except for the last three days of the experiment.

The first clinical signs of the development of LSD in cattle in bull No. 7 manifested themselves in the form of general weakness and hyperthermia on the sixth day after intravenous administration of the Privolzhsky strain (35 days of the experiment). Elevated body temperature in this individual was observed until the end of the experiment. In addition to fever, decreased appetite and lethargy, starting from the tenth day after infection, multiple painless nodules with a diameter of 1.0 to 5.0 cm were palpated on the skin in the area of the submandibular space, neck and shoulder blades of bull No. 7.

Full information about the results of thermometry of all animals used in the experiment is presented in figure 8.

Thus, the results of clinical observation of cattle showed that the strain "Neethling-ARRIAH" of the virus of infectious nodular bovine dermatitis is avirulent for naturally susceptible animals.

In addition, it was shown that this strain of bovine bovine skin disease virus causes the formation of protective immunity against infection with virulent forms of bovine skin disease virus.

The results of the pathological and anatomical autopsy after euthanasia of the animals showed that only 2 (bulls No. 5 and No. 7) out of 7 heads of cattle had visible signs of pathological changes in the internal organs.

Autopsy of individuals No. 5 and No. 7, in addition to skin lesions, revealed inflammatory processes in the prescapular lymph nodes and lymph nodes of the knee fold (erased border of the parenchyma and the cortical layer, hyperemia, an increase in size by 1.5-2 times compared to the norm). In addition, the development of dystrophic phenomena in the right kidney of the animal and extensive infiltration of the subcutaneous tissue with serous exudate were noted.

Thus, the data obtained during the pathoanatomical autopsy confirmed the results of clinical observation.

Samples of stabilized blood and nasal swabs taken within 28 days after the introduction of the strain "Neethling-ARRTAH" of the bovine itch virus showed the absence of the bovine itch virus genome in the blood and nasal swabs of all injected patients (No. 1, No. 2, No. 3, No. 4 and No. 6 ) and control (No. 5 and No. 7) animals.

The obtained results indicate the safety of the strain "Neethling-ARRIAH" of the infectious nodular bovine dermatitis virus for naturally susceptible animals and the environment.

Example 5. Immunogenic properties of the strain "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of cattle

To study the immunogenic properties of the strain "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of bovine animals, 20 heads seronegative to the virus of infectious nodular dermatitis of bovine animals were divided into 5 groups (4 heads in each).

Individuals of groups No. 1, No. 2, No. 3 and No. 4 (experimental groups) were immunized by subcutaneous injection into the region of the middle third of the neck of 1 cm ³ of the virus-containing material of the Neethling-ARRIAH strain of the infectious nodular dermatitis virus of cattle with biological activity:

Individuals of group No. 5 were not subjected to immunization (control group).

Within 10 days after immunization, clinical observations were made for all animals, including thermometry and inspection of the injection site.

21 days after immunization, the virulent strain "Privolzhsky" of the infectious nodular bovine dermatitis virus was titrated on the individuals of all five groups (according to the recommendations of the OIE). To do this, from vaccinated material of the specified strain with a cytopathic activity of 10 ^5.0 TCD ₅₀ /cm ³ (whole vaccinated material) by the method of serial dilutions was prepared by a series of tenfold dilutions (from 10 ^-1 to 10 ^-5 ). The obtained dilutions and the whole virus-containing material were administered intradermally to all animals in previously shaved areas on the right side. Each dilution and whole vaccinated material was administered in four repetitions of 0.1 cm ³ (Fig.9, Fig.10).

7 days after the titration in each animal of all groups, the development of a tissue reaction was recorded at the injection sites of dilutions and whole virus-containing material of the Privolzhsky strain of infectious nodular dermatitis virus. Visually, the reaction to the introduction of the specified strain developed in the form of edema; at the injection sites of the most concentrated viral material, the formation of small necrotic areas was recorded (Fig. 11, Fig. 12).

Based on the data obtained by the Reed and Mench method, the infectious activity titer of the Privolzhsky strain of infectious nodular bovine skin dermatitis virus was determined on each animal, after which the average titers of infectious activity of the Privolzhsky strain of infectious nodular bovine skin dermatitis virus were calculated for each group. The titer of infectious activity was expressed in ID ₅₀ .

After calculating the average titer of the infectious activity of the virulent strain by groups, the difference between the values of this indicator for the control group (group No. 5) and the experimental groups (groups No. 1, No. 2, No. 3 and No. 4) was determined.

The dose of the Neethling-ARRIAH strain is recognized as immunogenic against infection with virulent forms of infectious bovine skin rash virus if it provides the value of the difference between the mean titer of infectious activity of a virulent strain of infectious bovine skin disease virus on non-immunized and the average titer of infectious activity of a virulent strain of bovine skin disease virus on immunized animals not less than 10 ^2.5 ID ₅₀ .

The results obtained by the method of titration of the virulent strain of the virus of infectious nodular dermatitis of cattle on immunized with the Neethling-ARRIAH strain and on non-immunized naturally susceptible animals showed that all the doses of the Neethling-ARRIAH strain used in the experiment (10 ^4.85 TCD ₅₀ / cm ³ , 10 ^3.85 TCID ₅₀ /cm ³ , 10 ² ' ⁸⁵ TCID ₅₀ /cm ³ and 10 ^1.85 TCID ₅₀ /cm ³ ) are immunogenic and protect cattle from infection with virulent forms of the bovine LSD virus (table 3).

Example 6. Species of the strain "Neethling-ARRIAH" of the virus of infectious nodular dermatitis of cattle

The species affiliation of the Neethling-ARRIAH strain to the bovine nodular skin dermatitis virus was confirmed by the Sanger locus sequencing method (Applied Biosystems, USA). It is established that the strain "Neethling-ARRIAH" belongs to the group of viruses of infectious nodular dermatitis of cattle (Fig.13).

Sources of information taken into account when compiling the description of the invention to the application for the issuance of a patent of the Russian Federation for the invention "Strain" Neethling-ARRIAH "of the virus of infectious nodular dermatitis (lodular dermatitis) of cattle for the manufacture of biological products for the specific prevention of infectious nodular dermatitis of cattle":

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3. Immunobiological properties of the bovine lumpy skin disease virus strain - VND KRS/Dagestan/2015 / S.V. Kononova, O.P. Byadovskaya, A.V. Kononov, I.N. Shumilova, E.E. Artyukhova // Veterinary science today. - 2017. - No 2. - S. 58-61.

4. Cultivation of lumpy skin disease virus in cattle in transplanted lines of cell cultures / I.N. Shumilova, S.V. Kononova, B.L. Manin, N.V. Koropova, A.V. Kononov // Veterinary. - 2017. - No 7. - P.53-57.

5. Cultural and biological properties of the causative agent of lumpy skin disease in cattle isolated on the territory of the Russian Federation in 2015 / A.V. Kononov, S.V. Kononova, I.N. Shumilova, A.A. Nesterov, A.V. Shishkov, V.I. Diev, R.V. Yashin, A.V. Mishchenko // Veterinary science today. - 2016. - No 3. - P.8-18.

6. The problem of lumpy dermatitis in cattle / V.A. Mishchenko, A.V. Mishchenko, A.V. Kononov, V.N. Shevkoplyas, G.A. Jailidi, S.G. Dresvyannikova, O.Yu. Chernykh. // Veterinary science of Kuban. - 2015. - No 5. - P.3-6.

7. A lumpy skin disease virus which followed a recombination event demonstrates more aggressive growth in primary cells and cattle than the classical field isolate / S. Kononova, A. Kononov, I. Shumilova, O. Byadovskaya, A.A. Nesterov, P. Prutnikov, S. Babiuk, A. Sprygin // Transbound. Emerg. Dis. - 2021. - 68(3). P. 1377-1383.

8. Detection of vaccine-like strains of lumpy skin disease virus in outbreaks in Russia in 2017 / A.V. Kononov, O.P. Byadovskaya, S.V. Kononova, R. Yashin, N. Zinyakov, V. Mischenko, N. Perevozchikova, A. Spryg in // Arch, of Virol. - 2019. - Vol.164.-P. 1575-1585.

9. Determination of lumpy skin disease virus in bovine meat and offal products following experimental infection / A.V. Kononov, P.V. Prutnikov, I.N. Shumilova, S. Kononova, A. Nesterov, O. Byadovskaya, Y. Pestova, V. Diev, A. Sprygin // Transbound. Emerg. Dis. - 2019. - Vol.66. - P. 1332-1340.

10. Emergence of a new lumpy skin disease virus variant in Kurgan Oblast, Russia, in 2018 / A.V. Kononov, P. Prutnikov, O.P. Byadovskaya, S. Kononova, V. Rusaleev, Y. Pestova, A. Sprygin // Arch.of Virol. - 2020. - Vol.165. - P. 1343-1356.

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13. OIE Terrestial Manual - Paris, 2021. - Chap. 3.4.12.

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Claims (1)

The strain "Neethling-ARRIAH" of the virus of infectious nodular bovine dermatitis of the family Poxviridae of the genus Capripoxvirus, deposited in the All-Russian State Collection of Exotic Types of Foot and Mouth Virus Viruses and Other Animal Pathogens (GKSM) of the Federal State Budgetary Institution "ARRIAH" under registration number: No. 393 - dep./22- 27 - GKSHM FGBU "ARRIAH", for the manufacture of biological products for the specific prevention of infectious nodular dermatitis in cattle.

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