STRAIN FAdV / E / 04 / 2022 OF AVIAN ADENOVIRUS INFECTION VIRUS AVIADENOVIRUSHEPATITIDIS TYPE E FOR MANUFACTURE OF BIOPREPARATIONS FOR DIAGNOSIS AND SPECIFIC PREVENTION OF AVIAN ADENOVIRUS TYPE E

Abstract

FIELD: veterinary virology; biotechnology.SUBSTANCE: proposed strain FAdV / E / 04 / 2022 of the avian adenovirus infection virus Aviadenovirus hepatitidis species E of the Adenoviridae family, genus Aviadenovirus, is deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Virus and Other Animal Pathogens “Federal Center for Animal Health” (FSBI ARRIAH) under registration number No. 473-dep. / 23-23-GKShM of FSBI ARRIAH. The presented strain is reproduced in a primary trypsinized culture of chicken embryonic liver cells. Within 72 hours of incubation, the titer of infectious activity of the virus reaches 6.75±0.25 lg TCD50 / cm3.EFFECT: invention can be used to manufacture kits for diagnosing avian adenovirus type E and for monitoring the antigenic activity of vaccines against the disease caused by this virus.1 cl, 2 dwg, 6 tbl, 7 ex

Description

[0001] The invention relates to the field of veterinary virology and biotechnology, and can be used for the development and manufacture of diagnostic and specific prophylactic means for avian adenovirus type E.

[0002] Avian adenoviruses (FAdV) belong to the genus Aviadenovirus of the family Adenoviridae and are non-enveloped linear double-stranded DNA viruses. According to genetic and antigenic characteristics, 5 species (A, B, C, D, and E) are distinguished, including 12 serotypes (1-7, 8a, 8b, 9-11) of avian adenoviruses [1, 2].

[0003] FAdV are the causative agents of a number of infectious diseases in poultry of all ages. Chickens are particularly susceptible to adenoviruses, and infection can lead to pathologies such as inclusion body hepatitis, hydropericarditis, gastric erosion, and respiratory infections. Economic losses from adenovirus infections are due to high mortality among broilers, reduced growth rates and weight gain, decreased egg production, and the culling of meat products due to damage to the liver and other organs. Adenoviruses can act as primary pathogens or enhance pathogenicity when coinfected with immunosuppressive viruses, such as infectious bursal disease virus and infectious anemia virus. The spread of FAdV in commercial flocks is characterized by a high rate of horizontal transmission, virion resistance to environmental factors, and the possibility of vertical transmission [3, 4, 5, 6, 7].

[0004] Vertical transmission occurs at the beginning and end of the egg-laying period and occurs through virus penetration into the ovary or oviduct via the hematogenous or ascending route. Yolk infection results in embryonic death, while eggshell infection can result in latent infection of chicks, which manifests itself during immunosuppression. Infected birds exhibit delayed growth and development, increased susceptibility to other pathogens, and decreased productivity. Thus, vertical transmission complicates the control of adenovirus infections on poultry farms, as the virus circulates between generations of birds, leading to significant losses among young birds, especially if the chicks lack maternal antibodies to the virus [1, 2, 8].

[0005] Horizontal transmission occurs primarily via the fecal-oral route; the virus colonizes the intestinal epithelium within 12 hours of infection and can be detected in the blood within 24 hours. Adenoviruses reach the primary target organs, the liver and pancreas, within 2-3 days of infection. The duration of virus replication and shedding into the environment depends on the age of the bird and its immune status and can persist for several weeks to months, even in the absence of clinical signs. This is confirmed by recent studies where FAdV was detected in both healthy chickens and chickens with pronounced clinical symptoms [3, 4, 9, 10].

[0006] Recently, widespread FAdV transmission has been observed among broiler flocks in various countries, with a tendency toward endemic outbreaks rather than sporadic cases. Different strains of the same serotype differ in antigenic properties, pathogenicity, and virulence, which is due to genetic variations and can impact the clinical course of the disease, diagnosis, and vaccine development. Understanding these differences is critical for the effective control and prevention of diseases caused by avian adenoviruses [3, 7, 8, 9, 11].

[0007] Currently, FAdV strains isolated in the Russian Federation are known and used for the production of diagnostic and vaccine preparations.

[0008] The strain “ADV” of the avian hydropericarditis virus is known and is used in the production of antigen and immune serum for setting up a neutralization reaction for the purpose of laboratory diagnosis of avian hydropericarditis

[12] .

[0009] The strain "kr 95 No. 114 of the chicken hydropericarditis syndrome virus for vaccine production" belongs to serotype 4 of the adenovirus family. The strain was obtained through multiple sequential passages in sensitive biological systems and is used to produce vaccine preparations

[13] .

[0010] The strain "Biss No. 113" of the egg drop syndrome virus-76 is known, it belongs to the adenovirus species Duck adenovirus A. The strain was obtained by carrying out 4 consecutive passages on developing duck embryos, with the aim of using it for the production of vaccine preparations

[14] .

[0011] The "T-12" strain of the adenovirus inclusion body hepatitis-hydropericarditis virus (ADVH) is used for the production of diagnostics and vaccines and for experimental disease studies. This strain belongs to serotype 4 of adenoviruses and can be successfully cultivated in chicken embryo liver cell culture, on SPF and commercial embryos, and in the liver of 10-20-day-old chickens

[15] .

[0012] However, among the known strains, there is no strain suitable for the diagnosis and specific prophylaxis of avian adenovirus type E, which circulates in the Russian Federation. The significant genetic and antigenic diversity, combined with the lack of cross-immunity between adenovirus isolates of different species, makes it impossible to develop universal diagnostic and prophylactic agents. The objective of this invention is to expand the arsenal of serotype-specific adenovirus strains for the production of diagnostic kits and vaccines.

[0013] This problem was solved by obtaining a new strain “FAdV / E / 04 / 2022” of the avian adenovirus infection virus Aviadenovirus hepatitidis type E, which has high biological, antigenic and immunogenic activity.

[0014] The technical result of using the proposed invention consists in obtaining a new strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis type E, suitable for the manufacture of biological products for the diagnosis and specific prevention of avian adenovirus infection type E.

[0015] The specified technical result was achieved by obtaining the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis type E for the manufacture of biological products for the diagnosis and specific prevention of avian adenovirus type E.

[0016] The "FAdV / E / 04 / 2022" strain of avian adenovirus infection is new and previously unknown. The isolate of avian adenovirus type E was isolated at the All-Russian Research Institute of Animal Health from pathological material from 17-day-old broiler chickens exhibiting clinical signs of the disease.

[0017] To obtain the "FAdV / E / 04 / 2022" strain of avian adenovirus infection virus with optimal biotechnological properties, a primary trypsinized culture of chicken embryonic liver cells was used. A series of sequential passages in this cell culture yielded the "FAdV / E / 04 / 2022" strain of avian adenovirus infection virus (Aviadenovirus hepatitidis type E) from a previously isolated isolate. This strain has stable biotechnological properties and is suitable for the manufacture of diagnostic and prophylactic drugs for this disease.

[0018] Strain "FAdV / E / 04 / 2022" 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 registration number: No. 473 - dep / 23-23 - GKSHM of the All-Russian Research Institute of Animal Health.

[0019] The possibility of using the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis type E for the production of biological products for the diagnosis and specific prevention of avian adenovirus E has been experimentally confirmed.

[0020] The strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis type E is characterized by the following features and properties.

[0021] Identification and phylogenetic relationship of the avian adenovirus strain "FAdV / E / 04 / 2022". Identification was based on the determination of the primary structure of the Hexon gene of the test sample of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis species E, followed by an analysis of the phylogenetic relationship with other strains and isolates of avian adenovirus presented in the GenBank database.

[0022] The essence of the invention is reflected in the graphic images:

[0023] Fig. 1 – Dendrogram reflecting the phylogenetic relationships of strains and isolates of avian adenovirus type E based on a comparative analysis of the obtained Hexon gene (2844 nucleotides) with the complete sequences of this gene presented in the GenBank database;

[0024] Fig. 2 – Dendrogram reflecting the phylogenetic relationships of strains and isolates of avian adenovirus type E based on a comparative analysis of the obtained Hexon gene with incomplete sequences of this gene presented in the GenBank database (1085 nucleotides of the second half of the Hexon gene (position in the gene 1573-2657 nucleotides)).

[0025] The essence of the invention is explained by the following sequence lists:

[0026] SEQ ID NO:1 represents the 2844 nucleotide sequence of the avian adenovirus strain "FAdV / E / 04 / 2022";

[0027] SEQ ID NO:2 represents the 948 amino acid sequence of the avian adenovirus strain "FAdV / E / 04 / 2022".

[0028] To characterize the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis species E, the nucleotide and amino acid sequences of the Hexon gene were determined. Comparative analysis with previously published sequences using the BLAST program of the NCBI resource showed that the studied sequence (2844 nucleotides) with 100% nucleotide coverage (the complete Hexon gene) has the highest similarity of 99.19% with the sequence of the FV211-16 isolate of avian adenovirus type E from Peru (KX258422, FV211-16 / Broiler chicken / Peru / 2016 / serotype 8b / hexon gene), with 42% nucleotide coverage it has a similarity of 99.92% with the sequence of the FAdV / Broiler chicken / Kurdistan / Iraq / 2021 isolate of avian adenovirus type E from Iraq (MZ053469, sequence length 1219 nucleotides).

[0029] Isolate FV211-16 was isolated in 2016 in Arequipa, Peru, from liver samples from broiler chickens (2-4 weeks of age) with an outbreak (5-10% mortality) of inclusion body hepatitis (characterized by liver necrosis with microscopic eosinophilic or basophilic intranuclear inclusions in hepatocytes and a mortality rate of about 10%) caused by avian adenovirus. Phylogenetic analysis based on complete genome sequences showed that FV211-16 clusters with serotype 8b strains (KU517714, GU734104, and KT862811) with 95.4 to 97.1% identity, with the Malaysian strain UPM04217 being the most closely related.

[0030] The isolate FAdV / Broiler chicken / Kurdistan / Iraq / 2021 was isolated in 2021 in the city of Sulaymaniyah (Kurdistan Region, Iraq) from liver samples from broiler chickens (2-3 weeks of age) from an outbreak (mortality rate was 8-15%) of hepatitis with inclusion bodies accompanied by damage to the kidneys and pancreas caused by avian adenovirus.

[0031] Phylogenetic analysis confirms the relationship of the presented strain “FAdV / E / 04 / 2022” of the avian adenovirus infection virus Aviadenovirus hepatitidis species E with avian adenoviruses of species E and in particular with the isolate FV211-16 (Fig. 1) and with the isolate FAdV / Broiler chicken / Kurdistan / Iraq / 2021 (Fig. 2).

[0032] Morphological features

[0033] Strain "FAdV / E / 04 / 2022" belongs to the phylum Preplasmiviricota, class Pharyngeaviricetes, order Rowavirales, family Adenoviridae, genus Aviadenovirus, species Aviadenovirus hepatitidis (Fowl aviadenovirus E).

[0034] The virion has icosahedral symmetry, lacks an outer lipid envelope, and is 70-90 nm in diameter. The capsid consists of 720 hexon subunits formed by 240 trimers and 12 penton bases, each vertex bearing a fibril-like appendage protruding 20-30 nm from the particle surface. This capsid structure gives the virion its characteristic, distinct shape with distinct edges and vertices.

[0035] Viral replication, transcription and assembly of virions occur in the nucleus of the infected cell, where intranuclear basophilic inclusions characteristic of adenovirus infections are formed.

[0036] Antigenic properties

[0037] The "FAdV / E / 04 / 2022" strain of the avian adenovirus infection virus (Aviadenovirus hepatitidis species E) is characterized by pronounced antigenic activity and belongs to serotype 8b. The main viral antigenic determinants are localized in the capsid proteins, primarily in the hexon, pentyl base, and fibril protein.

[0038] The structural protein hexon contains conservative group-specific epitopes responsible for cross-immunological reactions between representatives of different serotypes of avian adenoviruses.

[0039] The serotype specificity of the "FAdV / E / 04 / 2022" strain of the avian adenovirus Aviadenovirus hepatitidis type E is determined by the variable regions of the fiber gene, which encodes a fibril protein that determines the virus's ability to bind to receptors on hepatocytes, endothelial cells, and cells of the reticuloendothelial system. This interaction ensures tissue tropism of the virus and induces the production of neutralizing antibodies.

[0040] The strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus has pronounced immunogenic activity due to the combination of conservative and variable epitopes in the capsid proteins, which contributes to the formation of a stable humoral immune response in susceptible birds.

[0041] Resistance to external factors

[0042] The "FAdV / E / 04 / 2022" strain of the avian adenovirus infection virus is stable in the external environment due to its dense protein capsid structure and the absence of a lipid membrane. The strain retains its infectious activity at room temperature for up to 8 weeks, at 4°C for up to 12 months, is inactivated within 7 days at 37°C, and within 30 minutes at 56°C. The strain withstands three freeze-thaw cycles without losing its antigenic properties. The strain is active in a pH range of 5.0-9.0; buffer media with a pH of 6.8-7.4 are optimal for storage; it is inactivated at pH 4.0 and 10.0. Among chemical agents, it is resistant to the effects of ether, chloroform and detergents, is completely inactivated when exposed to 1-2% formaldehyde for 24 hours at 20°C, and is sensitive to ultraviolet radiation.

[0043] Biotechnological properties

[0044] The "FAdV / E / 04 / 2022" strain of the avian adenovirus infection virus is characterized by pronounced replicative activity and high antigenic stability. The strain is adapted to cultivation on primary trypsinized culture of chicken embryonic liver cells at a titer of 7.75 log2. A pronounced cytopathic effect is formed 48 hours after infection and is characterized by cell rounding, the formation of intracellular inclusions, followed by lysis of the monolayer. The optimal cultivation condition is the use of a low-serum medium of 0.5%, at a temperature of 37°C for 72 hours. Experiments have confirmed that the biotechnological properties of the "FAdV / E / 04 / 2022" strain do not statistically change over 5 consecutive passages (observation period).

[0045] Additional features and properties

[0046] Pathogenicity – the strain has a moderate to high degree of pathogenicity, depending on the infecting dose, route of infection, and the immune status and age of the bird.

[0047] Virulence - highly virulent in chickens up to 6 weeks of age, causing mortality up to 30%, moderately virulent in adult birds, causing subclinical infection.

[0048] Stability – maintained for 5 consecutive passages in primary trypsinized chicken embryonic liver cell culture. Biological properties were characterized by determining the infectivity of the virus at each passage.

[0049] Contamination with bacteria, fungi, mycoplasma and foreign viruses - the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus is not contaminated with bacteria, fungi, mycoplasma and foreign viruses.

[0050] Storage conditions

[0051] The strain retains its biological properties when stored frozen for 12 months at -70±5°C. When lyophilized, the permissible shelf life is 10 years.

[0052] Geno- and chemotaxonomic characteristics

[0053] The strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus is a DNA-containing virus belonging to the genus Aviadenovirus, family Adenoviridae.

[0054] The viral genome is represented by a linear double-stranded DNA, 43.8 kb in length, with a molecular weight of about 28.5x10 6 Yes. At both ends of the genome are terminal inverted repeats, to the 5' ends of which a terminal protein is covalently attached, acting as a protein primer in the process of initiating viral DNA replication.

[0055] The genome of the avian adenovirus infection virus strain "FAdV / E / 04 / 2022" contains 47 open reading frames encoding structural and regulatory proteins, including the major capsid proteins and core proteins involved in DNA packaging.

[0056] The capsid has icosahedral symmetry (T=25) and consists of 250 capsomeres, including 240 hexon trimers and 12 penton bases, each of which carries a fibril backbone. The hexon is the main component of the capsid and contains variable loops L1, L2, and L4, which determine serotype specificity. The penton bases form the vertices of the icosahedron, to which fibril proteins are attached, participating in primary binding to host cell receptors and initiating enocytosis.

[0057] Chemically, the "FAdV / E / 04 / 2022" strain of avian adenovirus infection is a non-enveloped virus that does not contain lipids or carbohydrates. Proteins comprise 88% of the virion mass, and DNA accounts for 12% of the total mass. The primary molecular markers for genotyping and serological identification are the Hexon gene, which encodes the major capsid protein containing hypervariable loops, and the Fiber gene, which determines the virus's tropism for specific cell types, influencing virulence and the serotype-specific immune response.

[0058] Due to the dense protein capsid structure and the absence of a lipid membrane, the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus has high thermal stability and resistance to organic solvents, and is typically inactivated by exposure to 0.5% formalin for 24 hours at +37°C for representatives of the Adenoviridae family.

[0059] Figure 1 shows a dendrogram reflecting the phylogenetic relationships of strains and isolates of avian adenovirus species E based on a comparative analysis of the obtained Hexon gene with partial sequences of this gene presented in the GenBank database (1085 nucleotides of the second half of the Hexon gene (position in the gene 1573-2657 nucleotides)). Comparative analysis of the obtained nucleotide sequence showed that the strain isolated in Figure belongs to avian adenoviruses of species E.

[0060] According to the applicant, the proposed strain meets the patentability conditions of “novelty” and “inventive step”.

[0061] The essence of the proposed invention is explained by examples of its use, which do not limit the scope of the invention.

[0062] Example 1. Obtaining a viral suspension of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus.

[0063] Virus isolation from pathological material from birds and subsequent virus reproduction for the purpose of accumulation were carried out in a primary trypsinized culture of chicken embryonic liver cells (CELC). For this purpose, a suspension prepared from the pathological material was added to a monolayer of the primary trypsinized culture of chicken embryonic liver cells freed from the growth medium and exposed for 60 min at a temperature of (37.0 ± 0.5) ° C. Then, the PSP (or Eagle) maintenance medium with the addition of 0.5% fetal blood serum was added. The infected culture was incubated at a temperature of (37.0 ± 0.5) ° C for 72 h, then frozen at minus (70.0 ± 5.0) ° C, after thawing, it was centrifuged at 3000 rpm for 15 min. The resulting virus-containing material was used for subsequent passages. During the fourth passage after 72-hour incubation, pronounced replicative activity of the virus was observed, and optimal accumulation of viral material was achieved.Identification, consistency with the original isolate, and phenotypic stability were determined using PCR.

[0064] Thus, the strain “FAdV / E / 04 / 2022” of the avian adenovirus infection virus was isolated at the 4th passage of cultivation.

[0065] To determine the infectious titer of the virus, a 24-hour monolayer of primary trypsinized culture of chicken embryonic liver cells was used. The virus was diluted (from 10 -1 up to 10 -8 ) on PSP medium. Each dilution of the inoculum of the "FAdV / E / 04 / 2022" strain of the avian adenovirus infection virus was added in a volume of 1.0 cm 3per well of a 24-well culture plate with a monolayer of primary trypsinized chicken embryo liver cell culture. Four wells were infected with each dilution; an uninfected cell culture was used as a negative control; titration was performed in duplicate. Plates with the infected cell culture were incubated in a CO2 incubator in a humidified environment at 37°C. The final assessment of the virus titration results in CC PEC was performed 10 days after infection based on the cytopathic effect characteristic of FAdV / E (cell rounding and destruction of cytoplasmic outgrowths between cell islets). The titration results were considered reliable if the viability of CC PEC in the control uninfected plate was maintained. The titer of the infectious activity of the strain was calculated using the Kerber method as modified by Ashmarin and expressed as decimal logarithms per unit volume (lg TCD 50 / cm 3). The results of titration in 2 replicates are presented in Table 1. The infectious activity of the obtained strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus when titrated in a primary trypsinized culture of chicken embryo liver cells is 6.75±0.25 lg TCID 50 / cm 3 .

[0066] Example 2. Control of the stability of biological activity of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus.

[0067] The biological stability of the obtained "FAdV / E / 04 / 2022" avian adenovirus strain was tested in five consecutive passages, with the infectivity titer determined for each. Virus passage was performed in a primary trypsinized culture of chicken embryonic liver cells, with titration of each passage performed in duplicate. The results are presented in Table 2.

[0068] Thus, the isolated strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus exhibits stable biological activity during the next 5 consecutive passages in the primary trypsinized culture of chicken embryo liver cells, the titer of infectious activity of the strain is in the range of 6.75 - 7.58 lg TCID 50 / cm 3 .

[0069] Example 3. Obtaining the antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis species E.

[0070] To obtain the antigen of the avian adenovirus strain "FAdV / E / 04 / 2022", a viral suspension obtained as described in Example 1 was used. The virus-containing suspension was subjected to two freeze-thaw cycles and inactivated with a 0.3% ethyleneimine dimer solution. To remove ballast proteins, the suspension was clarified at 4330 g for 25 minutes. The viral suspension was first precipitated at 125300 g for 80 minutes, then the pellet was resuspended in PBS at a ratio of 10:1. In the next step, it was centrifuged through a layer of 30% sucrose at 125300 g for 60 minutes. The fraction containing the purified material was collected and diluted with PBS at a ratio of 1:3 and again precipitated for 2 hours at 125300 g. The resulting sediment was resuspended in PBS at a ratio of 100:1 of the initial volume.

[0071] The working dilution of the antigen was determined using an indirect ELISA. For this, two-fold dilutions of the FAdV-E antigen were prepared from 1:50 to 1:800 in 0.1 M carbonate-bicarbonate buffer, and 100 µl of each dilution were added to the wells of the plate. The plate was incubated at 4°C for 18 hours. After removing unbound antigen, 100 µl of a 1% BSA solution were added to the wells. The plates were incubated for 60 minutes at room temperature.

[0072] After this, the wells were washed three times with TBR-T buffer solution. 200 µl of control sera (negative and positive) at a dilution of 1:100 were added to the top horizontal row A, 100 µl of dilution buffer were added to the remaining horizontal rows and sequentially diluted in two steps from top to bottom from a dilution of 1:100 to 1:12800. After incubation for 30 minutes at a temperature of plus 37°C and washing, 100 µl of anti-chicken conjugate at a working dilution were added to all wells and incubated for 30 minutes at a temperature of plus 37°C. The plates were washed 3 times with TBR-T buffer solution. 100 µl of ABTS solution were added to each well of the plate and incubated for 10-15 minutes at room temperature. The reaction was then stopped by adding 100 µl of 1% SDS solution to each well. The antigen titer was defined as the last dilution at which the positive control serum had the highest titer, determined in the reaction with the standard control antigen.Double the final dilution of the antigen was considered the working dilution.

[0073] An antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis type E with a titer of 1:200 was obtained.

[0074] The obtained antigen was evaluated in ELISA using homologous and heterologous blood sera to avian adenoviruses, avian influenza (AI) viruses, infectious bronchitis (IB), infectious bursal disease (IBD), Newcastle disease (ND), infectious laryngotracheitis (ILT), infectious encephalomyelitis (IEP), egg drop syndrome-76 (EDS-76), reovirus (REO), metapneumovirus (MPV), mycoplasma gallisepticum (MG) and synovia (MS). The results are presented in Table 3.

[0075] Antibodies were detected only in positive chicken blood serum to FAdV-E (SPAFAS, USA); with heterologous sera, antigen activity did not exceed the background level (reaction with non-immune serum).

[0076] Thus, the antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis type E was obtained. The possibility of using the obtained antigen as a biopreparation for the manufacture of diagnostic kits was confirmed.

[0077] Example 4. Obtaining hyperimmune blood sera from chickens to the antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus Aviadenovirus hepatitidis species E.

[0078] To obtain hyperimmune blood serum from chickens, 40-day-old chickens grown from SPF chicken embryos that do not have specific antibodies to avian adenovirus were used.

[0079] For immunization, purified antigen from the avian adenovirus strain "FAdV / E / 04 / 2022" obtained as described in Example 3 was used. The antigen from the avian adenovirus strain "FAdV / E / 04 / 2022" was emulsified with an equal volume of Montanide ISA-70 adjuvant, manufactured by SEPPIC (France), in a 1:1 ratio. The resulting monovalent vaccine was administered into the pectoral muscle area in a volume of 1.0 cm 3twice with an interval of 14 days.

[0080] Fourteen days after the second immunization, blood samples were collected from the axillary vein of the chickens for indirect ELISA testing. To prepare hyperimmune serum, the chickens were bled. The resulting blood was stored in test tubes at 37°C. 0 The serum was incubated at 4°C for 30-60 minutes, then at 4°C for 24 hours. After 24 hours, the serum that had formed on the surface of the tube was carefully collected into sterile tubes. The serum was then inactivated in a thermostat at 57°C for 30 minutes.

[0081] The antibody titer and immunospecificity of the obtained serum were tested in an indirect ELISA using an antigen preparation from the avian adenovirus strain "FAdV / E / 04 / 2022", obtained as described in Example 3 and using kits for detecting antibodies to avian influenza (AI), infectious bronchitis (IB), infectious bursal disease (IBD), Newcastle disease (ND), infectious laryngotracheitis (ILT), reovirus (REO), metapneumovirus (MPV), mycoplasma gallisepticum (MG) and synovia (MS) produced by the Federal State Budgetary Institution "ARRIAH". Homologous reference serum was used as a positive control for the reaction, and negative (neutral) serum was used as a negative control. The study results are presented in Table 4.

[0082] Double immunization with the antigen preparation of the "FAdV / E / 04 / 2022" strain of avian adenovirus induces the production of antibodies in 40-day-old chickens with a titer of 5968. The immunospecificity of the obtained serum was confirmed using an indirect ELISA.

[0083] Thus, the claimed strain “FAdV / E / 04 / 2022” of the avian adenovirus infection virus can be used for the production of biopreparations for diagnostics and specific prophylactic agents.

[0084] Example 5. Detection of antibodies to avian adenovirus type E in field blood sera of chickens.

[0085] Detection of antibodies to FAdV type E was performed using an indirect ELISA method of twofold dilutions of blood serum. The antigen of the "FAdV / E / 04 / 2022" strain of the avian adenovirus infection virus, obtained as described in Example 3, was used as the antigen preparation. Hyperimmune serum obtained as described in Example 4 was used as a positive control. Sensitization of plates (Nunc, MaxiSorp, Denmark) with the antigen in 0.1 M carbonate-bicarbonate buffer (pH 9.5-9.6) was carried out for 18 hours at +4°C. A 0.05 M Tris-HCl buffer with 0.2 M NaCl containing 0.1% Tween-20 (pH 7.6) (TBS-T) and 1% bovine serum albumin (BSA) was used to dilute the test and control samples and the anti-species conjugate. A TBS-T buffer solution was used for interstage washing. The test and control sera were tested from a dilution of 1:100 to 1:12800. All components in the reaction were added in a volume of 100 µl and incubated at 37°C.To detect the formed complexes, a working dilution of anti-chicken immunoperoxidase conjugate from Synbiotics (USA) was used. ABTS solution was used as a substrate. The reaction was stopped by adding 1% sodium dodecyl sulfate (SDS) to each well. The reaction was monitored spectrophotometrically at 405 nm. Antibody titers were determined by the endpoint of the titration; titers of 400 and above were considered positive.

[0086] A study was conducted on 190 blood serum samples from chickens delivered from two poultry farms in the Russian Federation: egg (groups 1-6, 25 samples from six sites) and meat (groups 7-8, 20 samples from two sites), in which DNA of avian adenovirus type E was detected by PCR. The results of the study are shown in Table 5.

[0087] Specific antibodies to avian adenovirus E were detected in 77 of 150 serum samples from laying hens and in 4 of 40 serum samples from broiler chickens.

[0088] Thus, the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus can be effectively used for the production of biological products (antigen and positive control (hyperimmune serum)) for diagnostic kits and test systems.

[0089] Example 6. Obtaining the antigen of the strain "FAdV / E / 04 / 2022" of avian adenovirus on chicken embryos for the production of a vaccine.

[0090] Twelve-day-old SPF chicken embryos were used, placed in plastic cages with the air cell facing upward. These embryos were treated with a 1% iodine solution. Holes were punctured in the air cell area of ​​the prepared embryos with a sterile stylet to inoculate the virus.

[0091] The working dilution of virus-containing material was injected into the allantoic cavity using a Socorex automatic syringe with a 0.5 mm diameter, 15 mm long needle, in a volume of 0.2 cm³. After inoculation, the hole in the shell was sealed with a sterile wax-paraffin mixture.

[0092] Chicken embryos infected with the avian adenovirus strain "FAdV / E / 04 / 2022" were incubated for 7 days at a temperature of (37.5 ± 0.5)°C and a relative humidity of 50-70%. Infected embryos were ovoscoped daily. After incubation, the embryos were placed in a refrigerated chamber at a temperature of 2-8°C for 12-24 hours to prevent any minimal contamination of the allantoic fluid with red blood cells during collection.

[0093] Before dissection, the embryos were kept for 2-3 hours at room temperature until the condensation (moisture) on the shell evaporated.

[0094] The eggshell above the air cell was disinfected by flambéing and cut off along the air cell border with sterile, pointed scissors. Then, observing aseptic precautions, the shell membrane and underlying chorioallantoic membrane were opened with sterile tweezers, and the embryos were removed. The collected embryos were placed in a homogenizer with the addition of a sterile buffer solution at a ratio of 1:5 (tissue weight:buffer volume). The tissue was thoroughly homogenized until a homogeneous suspension was obtained. Confirmation of the presence of the "FAdV / E / 04 / 2022" strain of avian adenovirus E (proposed invention) in the resulting viral suspension was performed using PCR.

[0095] Thus, the antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus type E was obtained on chicken embryos for the production of vaccines in industrial quantities.

[0096] Example 7. Obtaining an experimental model of a vaccine based on the antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus.

[0097] Eight experimental samples were constructed to determine the optimal vaccine composition. Samples of the suspension used, obtained as described in Example 6, contained 5, 10, 15, 20, 25, 30, 35, and 40% tissue suspension of SPF chicken embryo carcasses infected with the antigen of the FAdV / E / 04 / 2022 strain of avian adenovirus E. All samples were inactivated with aminoethylethyleneimine dimer and adsorbed on 3% aluminum hydroxide gel in a 50÷50 ratio. Eight vaccine samples with different contents of the FAdV / E / 04 / 2022 strain antigen of avian adenovirus E were prepared.

[0098] The evaluation indicator for the selection of the optimal concentration of avian adenovirus E antigen in the composition of the inactivated vaccine and the determination of protective antibody titers against avian adenovirus E was the antibody titer in ELISA, which ensures protection of at least 80% of vaccinated chickens from challenge infection.

[0099] For the experiment, nine groups of 10 birds each, aged 5 days, were formed. Eight groups were immunized with vaccine samples Nos. 1–8 (groups Nos. 1–8, respectively). The inoculation volume was 0.5 cm³. Group No. 9 remained intact (the unvaccinated control). Before vaccination and then every 7 days, blood samples were collected from the birds to obtain serum and for antibody testing. The titer of antibodies to avian adenovirus type E was determined according to the “Methodological recommendations for the detection of antibodies to avian adenovirus type E by the enzyme immunoassay method when testing blood sera in one dilution”, Federal State Budgetary Institution “All-Russian Research Institute of Animal Health”, 2023. Before vaccination, the chickens were seronegative to avian adenovirus E. On the 28th day after immunization, a control infection was carried out with the strain “FAdV / E / 04 / 2022” of the avian adenovirus infection virus type E, obtained as described in Example 1. The viral suspension was administered intranasally at a dose of 6.25 lg TCID 50 / 0.5 cm 3The birds were clinically observed for 14 days after infection. The results are presented in Table 6.

[0100] Vaccine samples prepared using antigen-containing suspensions at 5, 10, and 15% concentrations failed to induce antibody production in vaccinated chickens throughout the experiment. Challenge infections of chickens in groups 1, 2, and 3 resulted in the majority of chickens becoming ill, manifested by severe depression, feed refusal, and diarrhea. Postmortem examination of internal organs revealed dystrophic liver lesions, and multiple inclusion bodies were detected in hepatocytes in prepared microscopic smears.

[0101] Vaccine samples #4 and #5, prepared from 20% and 25% suspensions, elicited a weak humoral response 21 days after immunization; no increase in antibody titers was observed by 28 days after immunization. Following challenge, the incidence rate in these groups was 80% and 50%, respectively, confirmed by pathological and histological examinations. Clinically healthy chickens did not exhibit the pattern described above, but isolated inclusion bodies were observed in liver smears.

[0102] The inclusion of 30, 35, and 40% tissue suspensions containing the antigen of the "FAdV / E / 04 / 2022" strain of avian adenovirus E in the vaccine ensured its high antigenic and immunogenic activity. Thus, by 28 days after administration, the preparation based on the 30% suspension induced the accumulation of antibodies to avian adenovirus E in chickens of Group No. 6, reaching a titer of 654±98, which protected 90% of the chickens in the group from a challenge with a live virus.

[0103] Increasing the suspension concentration to 35 and 40% (groups No. 7 and No. 8) enhanced the antigenic properties of the injected vaccine samples, ensuring the formation of antibody titers of 1078±131 and 1771±335, respectively, already on the 21st day after their administration, which continued to grow by the 28th day and amounted to 1872±107 and 2474±291 (Table 6). Preparations with such antigenic activity provided 90-100% protection of the immunized livestock from challenge infection after 28 days.

[0104] In group 9, 9 out of 10 infected chickens became ill after infection with the "FAdV / E / 04 / 2022" strain of avian adenovirus infection type E.

[0105] It has been shown that in order to form a minimum protective level of antibodies above 600 against avian adenovirus type E, it is necessary that the concentration of tissue suspension containing adenovirus E antigen in the vaccine preparation be at least 30%.

[0106] Thus, the obtained antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus E was used to produce a biopreparation for the specific prevention of avian adenovirus E. Consequently, the antigen from this strain is applicable for the creation of a vaccine that provides protection against avian adenovirus E.

[0107] Sources of information taken into account when preparing the description of the invention for the application for the issuance of a Russian Federation patent for the invention “Strain “FAdV / E / 04 / 2022” of the avian adenovirus infection virus Aviadenovirus hepatitidis type E for the manufacture of biological products for the diagnosis and specific prevention of avian adenovirus type E”:

[0108] 1. El-Shall NA, Abd El-Hamid HS, Elkady MF et al. Epidemiology, pathology, prevention, and control strategies of inclusion body hepatitis and hepatitis-hydropericardium syndrome in poultry: A comprehensive review. Frontiers in Veterinary Science. 2022; 9: 1075948. doi: 10.3389 / fvets.2022.1075948.

[0109] 2. International Committee on Taxonomy of Viruses (ICTV). Virus Taxonomy: 2023 Release. https: / / ictv.global / taxonomy

[0110] 3. Lebdah M., Alshaya D.S., Jalal A.S. et al. Molecular characterization of aviadenovirus serotypes and pathogenicity of the identified adenovirus in broiler chickens. Poultry Science. 2022; 101(12): 101918.

[0111] 4. Reece R.L., Grix D.C., Barr D.A. An unusual case of inclusion body hepatitis in a cockerel. Avian Diseases. 1986; 30: 224-227. doi: 10.2307 / 1590640.

[0112] 5. Schachner A., Marek A., Jaskulska B. et al. Fowl adenovirus biology and pathogenesis. Veterinary Microbiology. 2018; 215: 34-44.

[0113] 6. Hess M. Adenoviral infections in poultry: A review. Avian Pathology. 2000; 29(5): 403-413.

[0114] 7. Niczyporuk J.S., Samanta J. et al. Molecular characteristics of fowl adenovirus strains detected in broiler chickens on diets without immunostimulant supplements. Journal of Veterinary Research. 2024; 68(2): 207-214. doi:10.2478 / jvetres-2024-0028.

[0115] 8. Butcher G.D., Hossiny M. Fowl Adenovirus Hepatitis (IBH and HHS): An Emerging Poultry Disease. Veterinary Medicine (EDIS). 2024; Vol. 2024 No. 4. doi:10.32473 / edis-vm263-2024.

[0116] 9. Ugwu C.C., Mohd Hair-Bejo, Nurulfiza M.I. et al. Efficacy, humoral, and cell-mediated immune response of inactivated fowl adenovirus 8b propagated in chicken embryo liver cells using bioreactor in broiler chickens. Veterinary World. 2022; 15(11): 2681-2692.

[0117] 10. Grafl B., et al. Infectious diseases caused by FAdV in poultry. Avian Pathology. 2012; 41(5): 489-496.

[0118] 11. Lu H., Wang W., Zhang J. et al. An efficient fiber-based ELISA for detection of antibody against fowl adenovirus serotypes 7 and 8. Journal of Veterinary Diagnostic Investigation. 2020; 32(3): 444-449.

[0119] 12. RF Patent No. RU 2120993 C1, IPC C12N7 / 00, 1998

[0120] 13. RF Patent No. RU 2193062 C1, IPC C12N7 / 00, 2002

[0121] 14. Russian Federation Patent No. RU2221865C1, IPC C12N 7 / 00, 2004.

[0122] 15. RF Patent No. RU 2 277 584 C1, IPC C12N7 / 00, 2006.

[0123] Table 1

[0124] Determination of the infectious activity of the strain "FAdV / E / 04 / 2022" by titration in the QC PEC

[0125] Titration frequency Virus propagation Virus titer, lg TCID 50 / cm 3 10 -3 10 -4 10 -5 10 -6 10 - 7 10 -8 1 ++++ ++++ ++++ ++++ +--- ---- 6,75 2 ++++ ++++ ++++ +++- ++-- ---- 6,75

[0126] Note: “+” – presence of cytopathic action;

[0127] «-» – absence of cytopathic effect.

[0128] Table 2

[0129] Stability of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus type E during reproduction in a primary trypsinized culture of chicken embryonic liver cells

[0130] n=2

[0131] Passage number Titration frequency Virus propagation Virus titer (lg TCID 50 / cm 3 ) Average virus titer (lg TCID 50 / cm 3 ) 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 5 1 ++++ ++++ ++++ ++++ +--- ---- 6,75 6,75 2 ++++ ++++ ++++ +++- ++-- ---- 6,75 6 1 ++++ ++++ ++++ ++++ +++- ---- 7,0 7,12 2 ++++ ++++ ++++ ++++ +--+ ---- 7,24 7 1 ++++ ++++ ++++ ++++ +++- ---- 7,33 7,17 2 ++++ ++++ ++++ +++- ++-- ---- 7,0 8 1 ++++ ++++ ++++ ++++ +++- ---- 7,33 7,12 2 ++++ ++++ ++++ ++++ +++- ---- 7,33 9 1 ++++ ++++ ++++ ++-+ +--- ---- 7,5 7,58 2 ++++ ++++ ++++ ++++ +--- ---- 7,66

[0132] Table 3

[0133] Test results of the antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus type E (proposed invention) using homologous and heterologous blood sera by the ELISA method

[0134] № Sample characteristics Antibody titer* S / P Qualitative assessment of the result 1 Chicken blood serum to the GP virus (Zoetis, USA) 0 0,00 negative 2 Chicken blood serum to IBV virus (Zoetis, USA) --- --- negative 3 Chicken blood serum to IBD virus (Zoetis, USA) 195 0,15 negative 4 Chicken blood serum to ND virus (Zoetis, USA) 155 0,13 negative 5 Chicken blood serum to ILT virus (Zoetis, USA) 2 0,01 negative 6 Chicken blood serum to the IEP virus (Zoetis, USA) 14 0,03 negative 7 Chicken blood serum to reovirus (Zoetis, USA) 7 0,02 negative 8 Chicken blood serum to MG (Zoetis, USA) 239 0,17 negative 9 Chicken blood serum for MS (Zoetis, USA) 32 0,05 negative 10 Chicken blood serum to metapneumovirus (IZSdV, Italy) 155 0,13 negative 11 Chicken blood serum to the EDS-76 virus (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 70 0,08 negative 12 Normal chicken blood serum (Zoetis, USA) 1 0,004 negative 13 Positive chicken blood serum to FAdV-E (SPAFAS, USA) 7456 1,47 positive 14 Positive chicken blood serum to FAdV-A (SPAFAS, USA) 338 0,21 doubtful 15 Positive blood serum of chickens to FAdV-B (SPAFAS, USA) 449 0,25 doubtful 16 Positive blood serum of chickens to FAdV-C (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 85 0,09 negative 17 Positive blood serum of chickens to FAdV-D (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 32 0,05 negative

[0135] Note: "---" - negative result;

[0136] * - antibody titer is the reciprocal of the serum dilution

[0137] Table 4

[0138] Test results of hyperimmune blood serum of chickens to the antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus type E (proposed invention)

[0139] № Kit Antibody titer* Qualitative assessment of the result 1 Antigen of the strain "FAdV / E / 04 / 2022" 5968 positive 2 A kit for the detection of antibodies to the avian influenza virus by the enzyme immunoassay method when testing sera in a single dilution (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 0 negative 3 A kit for the detection of antibodies to the chicken infectious bronchitis virus by the enzyme immunoassay method when testing sera in a single dilution (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 103 negative 4 A kit for the detection of antibodies to the infectious bursal disease virus by the enzyme immunoassay method when testing sera in a single dilution (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 365 negative 5 A kit for the detection of antibodies to the Newcastle disease virus by the enzyme immunoassay method when testing sera in a single dilution (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 159 negative 6 A kit for the detection of antibodies to the avian infectious laryngotracheitis virus by the enzyme immunoassay method when testing sera in a single dilution (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 162 negative 7 A kit for the detection of antibodies to the causative agent of reovirus infection in birds using the enzyme immunoassay method when testing sera in a single dilution (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 272 negative 8 A kit for the detection of antibodies to avian pneumovirus by the enzyme immunoassay method when testing sera in a single dilution (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 84 negative 9 A kit for the detection of antibodies to Mycoplasma gallisepticum by the enzyme immunoassay method when testing sera in a single dilution (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health") 299 negative

[0140] Note: "---" - negative result;

[0141] * - antibody titer - the reciprocal of the serum dilution

[0142] Table 5

[0143] Detection of antibodies to the antigen of the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus type E in field blood sera of chickens

[0144] Group number Bird age (days) Total number of samples Number of positive samples Average antibody titer per group ±standard error of the mean 1 365-368 25 25 6176±767 2 365-368 25 17 1048±293 3 365-368 25 21 1056±204 4 183-186 25 4 244±50 5 183-186 25 8 240±34 6 183-186 25 2 164±17 7 37 20 2 130±21 8 38-40 20 2 170±48

[0145] Table 6

[0146] Antigenic and immunogenic activity of vaccine samples depending on the concentration of avian adenovirus E antigen

[0147] n=10

[0148] Group No. Number of chicks in a group Antigen concentration in the vaccine, % Antibody titers in ELISA / days after vaccination Number of sick chickens* 7 days 14 days 21 days 28 days 1 10 5 136±23 149±24 172±20 162±31 10 2 10 10 179±14 152±11 164±37 159±47 9 3 10 15 142±26 138±42 201±74 253±61 9 4 10 20 154±31 157±25 341±121 337±115 8 5 10 25 148±17 164±43 572±112 589±232 5 6 10 30 176±39 143±21 475±102 654±98 1 7 10 35 164±43 576±87 1078±131 1872±107 1 8 10 40 153±33 454±112 1771±355 2474±291 0 9 10 Control 157±47 174±63 169±26 182±32 9

[0149] Note: * - the number of sick chickens after control infection with the strain "FAdV / E / 04 / 2022" of the avian adenovirus infection virus type E.

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