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The study examines the DNA transformation dynamics of ВНК-21/SUSP/ARRIAH subline cells during rabies virus reproduction. Cells infected with the virus and control intact cells were cultivated under similar conditions. The identification of dependence of the virus infectivity on reproduction time revealed that the virus infectivity titre increased from (3.2 ± 0.2) lg CCID50/cm3 at the time of inoculation to (7.63 ± 0.3) lg CCID50/cm3 after 48 hours of reproduction, with the most intensive increase having been observed within the first 24 hours. The cell concentration changed from 0.5 to 1.9 million/cm3, i.e. increased by a factor of 3.8. After 24 hours, the cell growth rate slowed down. Findings from the examination of cell cycle phases during rabies virus reproduction in the host cell allowed for the estimation of duration and predominance of G1, S, G2 + M phases at different stages of cultivation. The dynamics of changes in the apoptotic cell population in the control and test samples was similar within 36 hours of cultivation. After the said period, the proportion of apoptotic infected cells was 28–42% higher than that of apoptotic control cells. After 9 hours, the proportion of cells undergoing G1 phase increased by 11.7% in the test samples, whereas it decreased by 16.6% in the control samples. Subsequently, the number of G1 phase cells in the control and test samples changed in the same way: a 40% decrease was observed after 15–18 hours, it was followed by a 45–46% growth jump, then again a 39–40% decrease and an increase were observed. After 33 hours of reproduction and till the end of cultivation, the proportion of infected cells undergoing G1 phase was significantly higher (by 12–21%) as compared with control cells. The percentage of S phase cells in the test and control samples was the same during the first day of the virus reproduction, with sharp jump-like 3.4- and 2.4-fold increases having been observed after 15 and 24 hours, respectively. After 24 hours, the infected and control cells began to demonstrate differences, which gradually increased from 8 to 137% by the end of reproduction. After 30 hours of reproduction, the proportion of test sample cells undergoing G2 + M phase began to decrease by 17–28% as compared with the control cells. The cell switch-over to the synthesis of complete rabies virus particles occurred after 24 hours of reproduction. This is indicated by changes in the host cell cycle phases, as well as by the slowing down of ВНК-21/SUSP/ARRIAH cell population growth.

Currently fluorescent quantitative real-time polymerase chain reaction, which is a cutting-edge technology in genetic diagnosis, is used in different areas of molecularbiology. Practicaladvantageofsimplicityaswellascombination of high speed, sensitivityandspecificitymadeitpossibletousethisanalysisfor nucleic acidquantitation. Thepaperpresentsgeneral information andrecommendedrules for thedevelopmentof real-timeqPCR. Thepublication isaimedtoacquaint theresearchersandreviewerswith necessaryrequirementstobefollowedin order toensure high accuracy, reliabilityandtransparencyof theexperiments, correct interpretation andrepeatabilityof thetest results. Currentapproachesaredescribedthatallow obtainingreliableandconsistent resultsbydifferentoperators, at differenttimesandin differentlaboratories. Basicrequirementsforreagentsused, nucleotidesequencesandvalidationmethodsaregiven. In general, thepublication givestheinformation neededtoachievethreeultimategoals: toprovidetheauthorswith abroadrangeoftoolsandrequirementsforthedevelopmentof real-time qPCR based-techniques; togivethepossibilitytothereviewersandeditorsofassessingthequalityofarticlesandguidelines/instructions in accordance with the requiredcriteria; toobtain consistentandreliableresultsof testsperformedusingthis method.

Inactivation is the loss by a virus of ability to reproduce and infect susceptible animals while retaining its antigenic properties. In this paper, the effectiveness of aminoethylethanolamine when used as an FMDV inactivant is shown. The inactivation rates under selected parameters, effect of aminoethylethanolamine on virus stability during inactivation and on vaccine immunogenicity after storage at 2–8 °С were determined. The method for calculation of 50% aminoethylethanolamine inactivating concentration (IC50) which enables to determine quality parameters of the virus-containing suspension, to compare the inactivating agent activities and their ability to ensure the vaccine innocuity within the given period of time is presented. It was established that IC50 for purified and non-purified virus-containing suspensions was identical (0.0045%), and its safety after 12 h of inactivation was one TCID50 per 109–1011 L of the virus containing suspension. It was also found that double increase in inactivation time increased the virucidal activity of aminoethylethanolamine by a factor of 1.8 for serotype O and 2.4 for serotype A. At the same time, the removal of cell debris had no significant effect on the inactivation process. Aminoethylethanolamine does not destroy 146S virus particles and it was confirmed by immunogenicity testing of the vaccines during storage. This means that 15% aqueous solution of aminoethylethanolamine, manufactured by Russian Company OOO “BIOKHIMRESURS” (Vladimir) complies with high quality standards. Immunogenicity test of bivalent FMD vaccine for cattle by challenging demonstrated that its potency was 10.08 protective doses per 2 cm3 of the vaccination dose.

Rabies is a dangerous zoonoticdisease that affects the central nervous system, causes encephalomyelitis and paralyses and Is almost invariably fatal. The disease causes significant economic losses associated with the death of animals, outbreak consequences, strict restrictions on domestic and international trade in livestock products, preventive and quarantine measures, laboratory tests. The World Organization for Animal Health recommends vaccination to control rabies. Taking into account that there is a lack of affordable high-quality vaccines to globally prevent and control the disease, stable, attenuated production strains of rabies virus with broad cross-activity against various variants of the pathogen shall be considered as ideal candidates to produce high-quality, safe and effective vaccines. Currently, someapproachesareappliedtoreducethevirusvirulenceandimprovesafetyof rabies vaccines. Reverse genetics is very popular now. It provides new approaches to study functions of a specific gene by analyzing phenotypic effects after direct manipulations with nucleotide sequences. The methods of reverse genetics have revolutionized molecular biology and have become apowerful tool to study genetics of RNA viruses. These methods are widely used to study rabies virus. The use of reverse genetics has made it possible to modify rabies virus production strains for manufacture of modern genetically modified rabies vaccines that induce a persistent and long-term immunity. The review briefly covers general approaches to development of viral vectors with the purpose to create genetically modified rabies vaccines.

In vaccine production, it is particularly important to purify the virus-containing suspension in order to remove ballast proteins and fats, which, when present in high concentrations, are responsible for depression or allergic reactions in animals. Polyguanidine and its derivatives have long been used for such purposes. At present, the market offers polyhexamethylene guanidine hydrochloride, a cationic polyelectrolyte with aunique combination ofphysico-chemical and biocidal properties allowing for it to be used in nearly all spheres of economy. Flocculation properties of polysept (polyhexamethylene guanidine hydrochloride) vary from batch to batch, and this has necessitated the development of a test system for determination of the incoming material quality, which has a significant impacton virus antigen concentration during vaccine production. Seven batches of polyhexamethylene guanidine were tested for flocculation properties, changes in FMDV immunogenic component concentration in the virus-containing suspension, osmolality of solutions at different percentage concentrations. Indicators of incoming material suitability for FMD vaccine production were determined. The batches of polysept should be tested for flocculation properties at different concentrations of thepolymer (0.007, 0.0105 and 0.01575%) in dynamics during 24 hours. After this period, the turbidity of solutions should not exceed 30 FNU (formazin turbidity) at concentrations of 0.0105 and 0.01575%. It is also necessary to determine the osmolality of polysept solutions at different percentage concentrations (6, 8, 10, 12, 14%). Osmolality values should be within the following ranges: 260 ± 20 mOsm fora 6% solution; 330 ± 25 mOsm foran 8% solution; 400 ± 25 mOsm fora 10% solution; 460 ± 30 mOsm fora 12% solution; 520 ± 20 mOsm fora 14% solution.

The results of the studies of cytomorphological, karyological, cultural properties of continuous suspension ВНК-21/SUSP/ARRIAH subline of newborn Syrian hamster kidney cells intended for foot-and-mouth disease, rabies, bovine parainfluenza-3, Aujeszky’s disease virus reproduction, as well as for production of diagnostic veterinary biologicals are presented. When cultured in suspension, BHK-21/SUSP/ARRIAH cell subline undergoes selection towards hypoploidy: modal class is represented by cells with 41 chromosomes (32–40% of cells); the share of cells containing 40–42 chromosomes is 78–80%; the share of polyploids averages around 1%; the range of variation in the number of chromosomes is from 36 to 54. BHK-21/SUSP/ARRIAH cell subline cultured in suspension with cell seeding concentration of 0.6–0.8 million cells/cm3 demonstrates growth rate of 6.67–11.00 and 96–99% cell viability. After 48 hours, G1-phase (diploid-2n) cells prevail in the cell population of the new subline (30.0–75.0% of cells); cells that undergo preparation for mitosis (S-phase) and mitosis (G2+M-phase) account for 3.0 to 20.0% of the entire population; the number of meganucleated and multinucleated cells (>4n) at the beginning and at the end of the logarithmic phase increases to 2%. BHK-21/SUSP/ARRIAH cells recover rapidly after cryopreservation and demonstrate 95–99% viability and growth rate of 3.36–5.88 at passages 1 to 3 and 6.85–10.95 at passages 4 to 12. Continuous suspension BHK-21/SUSP/ARRIAH cell line ensures virus accumulation at the following titres: FMD virus – 7.30– 8.00 lg TCID50/cm3, rabies virus – 7.25–8.00 lg CCID50/cm3, bovine parainflunza-3 virus – at least 6.00 lg TCID50/cm3, Aujeszky’s disease virus – 7.50–7.80 lg TCID50/cm3.

Compliance with the existing purity and safety requirements for immunobiologicals can be effectively achieved by the use of serum-free nutrient media and specialised supplements of non-animal origin. The paper shows the possibility of using Sheff-Vax ACF® supplements (Kerry, Inc., Ireland) for ВНК-21/SUSP/ARRIAH cell cultivation and FMDV reproduction. By passage 7, cell concentration and growth rate with Sheff-Vax Plus PF ACF were found to be 40–60% higher than with Sheff- Vax PF ACF and Sheff-Vax Plus ACF. No differences were observed as regards changes in pH. During FMDV reproduction in the cells, it was found that the number of 146+75S components in the test samples containing 1 million cells was 2.3–2.4 higher compared to the controls. Cells cultured with the use of Sheff-Vax Plus PF ACF supplement had normal morphology and multiple dynamic protrusions. In the presence of this supplement, growth rate and suspension concentration in the test and control samples became equal by passage 7. The number of immunogenic components of FMDV reproduced in the cells grown using Sheff-Vax Plus PF ACF was 20–30% higher than in the cells grown using other supplements. ВНК-21/SUSP/ARRIAH cell concentration and growth rate in the presence of specialised supplements were found to be lower than those in the control samples with serum and blood protein hydrolysate added to the nutrient medium. The virus yield from 1 million cells was higher in the culture grown using Sheff-Vax ACF supplements. Sheff-Vax Plus PF ACF was found to be the most suitable for ВНК-21/SUSP/ARRIAH cell cultivation and FMDV reproduction in the said cells out of the three tested supplements.

Foot and mouth disease has a negative impact on economy due to the high cost of eradication campaigns and stringent measures imposed on domestic and international trade in animal products. Prevention and control measures include mass vaccination of susceptible animals and control of post-vaccination immunity level. Concentration of 146S particles, which are the main component affecting the vaccine immunogenicity, is determined during commercial scale production of FMD vaccines. The paper assesses feasibility of spectrometric analysis for indirect determination of 146S component concentration while measuring amount of FMDV RNA isolated after serological binding. This method is cheap, easy-to-use and makes it possible to determine indirectly concentration of FMDV 146S particles in inactivated vaccine raw materials within 3–4 hours. Study of cultural FMDV suspensions shows that the linear model С146S = (3.9 × NRNA 146S + 566,783,689)/ 280,818,944,837 makes it possible to estimate FMDV 146S component concentration in the vaccine raw materials with the help of a spectrometric analysis. The actual results obtained in real-time reverse transcription – polymerase chain reaction (rtRT-PCR) were 97.0–99.9% consistent with the expected results of the spectrometric analysis used to determine cultural FMDV 146S component concentration. When compared to a complement fixation test, the actual results were 94.5–99.5% in line with the expected ones. The actual results for positive control were 99.0–99.6% in line with the expected ones. As expected, no FMDV genome or 146S particles were detected in the negative control sample.

During FMD vaccine production, special attention is paid to the concentration of 146S particles bearing the critical biological features of FMDV and being the main components that have an effect on vaccine immunogenicity. For this reason, each batch of vaccine raw material is tested for 146S component concentration. The paper presents the results of the use of a spectrometric method for whole particle concentration determination during quantification of FMDV RNA extracted after immune capture. It is an inexpensive, easy-to-perform method allowing for determination of FMDV 146S particle concentration in the non-inactivated culture suspension. 146S particle concentration was found to depend on the number of RNA molecules extracted from virions after their strain-specific immune capture and quantitatively detected by the spectrometric method. The presented method allows for determination of 146S component concentration in the non-inactivated vaccine raw material using the proposed linear model. The spectrometric method showed 94.5–99.5% correlation with real time reverse transcription polymerase chain reaction and complement fixation test based on the results of tests of 360 non-inactivated suspensions of FMDV of all types. Tests of the positive control demonstrated 99.0–99.6% compatibility of actual and expected results. FMDV genome and 146S particles were not detected in the negative control, and that was in line with expectations.

During reproduction in biological systems, FMD virus forms four variants of components, three of which do not include RNA of the virus. In the process of industrial production of FMD vaccines, special attention is paid to the number of whole virions, which have the most important biological properties of FMD virus and are the main components that determine the immunogenicity of vaccine preparations. Raw materials for vaccines at various stages of the technological process are tested for concentration of FMDV 146S component. The traditional method of determination is quantitative complement fixation test. In recent years, real-time RT-PCR has been used for indirect determination of FMDV 146S component concentration in a virus-containing suspension. The article presents a new approach to indirect determination of FMDV 146S component concentration in a non-inactivated suspension by comparing the maximum extreme points of the graphs of the second derivative of the fluorescence signal accumulation curves and the number of amplification reaction cycles. The dependence between FMDV 146S component concentration and the maximum extreme points of the graphs of the second derivative of the fluorescence signal accumulation curve is presented in the form of a square function: C146S FMDV = 0.0111(Cp)2 – 1.0157Cp + 20.446 with a high accuracy of approximation (R2 = 0.993). The proposed model allows to quantitatively estimate the content of 146S component in virus-containing vaccine raw materials. The presented method allows studying a large number of samples of non-inactivated raw materials for FMD vaccine in 4–5 hours. The main advantage of the proposed method is the capacity to determine the concentration of FMDV 146S component in a suspension with a high level of ballast proteins (more than 7.00 mg/cm3) and complete viral particles (from 0.01 to 5.00 μg/cm3).

FMD risk in the Russian Federation dictates the need for enhanced measures aiming to prevent the introduction of FMD virus and comprising systematic monitoring research and mass vaccination of susceptible animals in the buffer zone. Research into the development of vaccines for early protection confirm that their use induces the formation of virus-neutralizing antibodies in naturally susceptible animals in the outbreak area, which protects from FMD infection, limits its spread and contains it within the primary outbreak. Taking into account the high speed of the infection spread, such a control measure as using FMD vaccines which induce early protection should be adopted immediately after the occurrence of the outbreak. The article presents the results of the research into the formation of humoral immunity in naturally susceptible animals triggered by administration of inactivated emulsion FMD vaccines capable of ensuring early protection against type O FMD. Culture FMD virus of О/Primorsky/2012, О/Saudi Arabia/08 and О/Mongolia/2017 strains was used for vaccine production. Immunogenic activity of vaccines was tested in cattle, pigs, and sheep. It was found that monovalent emulsion FMD vaccine based on О/Mongolia/2017 strain induced the formation of virus-neutralizing antibodies in the quantity necessary to protect against the homologous strain in seven days after a single administration in the dose of 2 cm3 . Vaccines based on О/Saudi Arabia/08 and О/Primorsky/2012 FMDV strains can protect animals from infection with heterologous О/Mongolia/2017 strain at early stages if a double dose is administered. Vaccines based on the above-mentioned strains induce early immunity formation (seven days after vaccination) against type O FMD. We suggest using the given products in the zones of a higher risk of the virus introduction.

At present, Miramistin drug (benzyldimethyl [3-(myristoylamino) propyl] ammonium chloride monohydrate), which has a broad bactericidal effect, is used in veterinary practice. This antiseptic is active against most viruses, mycoplasmas, bacteria, fungi and protozoa. According to toxicometric parameters, Miramistin is classified as a low-hazard substance. Use of cell structures as test systems for assessing the toxicity of pharmacological substances instead of conventional tests on experimental animals allows us to better clarify the possible mechanism of the effect of the investigated substance. Since many types of mycoplasmas affect the genitourinary system organs, to assess the cytotoxicity of Miramistin kidney mammalian cells of various mammals can be used as test systems, in particular, the newborn Syrian hamster (VNK-21), Siberian mountain ibex (PSGK-30), and others. The possibility of using BHK-21 and PSGK-30 cell monolayer as test systems for assessing the baseline cytotoxicity of Miramistin was shown. When studying toxicity of the drug, the effect of its various concentrations on the cell morphology was studied, the number of viable cells and the total protein content were determined as an indicator of the cell mass increase. The results of a cytomorphological study indicate that the Miramistin maximum permissible concentration of for BHK-21 and PSGK-30 cell monolayer is 25 μg/cm3. The use of this antibacterial drug in higher concentrations caused the appearance and increased signs of endogenous intoxication and degeneration. When evaluating the proliferative activity of cells under the influence of the Miramistin antibiotic in different concentrations, it was found that increasing the dose to 50, 75, 100, 125, 150 μg/cm3 leads to decrease in the rate of cell growth compared to the control. The Miramistin content in the growth medium in an amount of up to 25 μg/cm3 did not affect the intensity of protein synthesis. Presence of Miramistin in the culture medium at the maximum permissible concentration causes a slight decrease in the reproduction of foot and mouth disease virus in BHK-21 and PSGK-30 cell cultures by 4.5 and 4.0%, respectively, compared with the control without antibiotic. Since mycoplasmas are the most common contaminants of cell cultures, further studies will be aimed at exploring the possibility of using Miramistin for decontamination of BHK-21 and PSGK-30 cell monolayers.

Rabies is one of the most important human and animal viral diseases, being one of the most dangerous zoonoses, causing damage to the central nervous system with an inevitable fatal outcome. This disease is of global concern, and it attracts special attention of international organizations (WHO, OIE, FAO, GARC) and of veterinary services in many countries around the world. A variety of anti-rabies vaccines have been used for specific rabies prevention in wild carnivores, however, the safety and effectiveness of some of them is doubtful. New, more advanced products are being developed, one of which is Ferarabivac, a live oral vaccine. The vaccine was tested for its immunogenicity and protectivity in wild carnivores. The optimal immunizing dose was 2.0 cm3, with the infectivity titre of RV-97 strain of at least 6.00 lg KKID50/cm3. Anti-rabies antibody titres detected in the blood sera of foxes and raccoon dogs 14 days post vaccination, were 0.70 ± 0.18 and 0.73 ± 0.19 IU/cm3, respectively, which provided protection against rabies virus infection (≥ 0.50 IU/cm3). Rabies virus neutralizing antibodies in foxes reached their maximum level of 4.30 ± 0.32 IU/cm3 50 days post vaccination. Antibody titres in vaccinated raccoon dogs also reached their maximum level of 4.53 ± 0.27 IU/cm3 50 days post vaccination. The minimum protective threshold levels of serum neutralizing antibodies was determined 12 months after the vaccination, and it was 0.62 ± 0.28 and 0.71 ± 0.17 IU/cm3 in foxes and raccoon dogs, respectively, which proves the necessity to perform booster vaccination one year later. No animals vaccinated against rabies with Ferarabivac live vaccine showed any clinical signs of the disease during the entire observation period following the challenge test carried out 30 days post vaccination.

Despite all the efforts made, the issue of rabies in the world does not lose its relevance. As an acute endemic infection, it poses a considerable danger to both animals and humans. The leading role in the transmission of rabies to humans belongs to diseased dogs; stray animals can be potential sources of rabies agent, which increases the risk of transmitting a deadly virus to humans. Rabies prevention in dogs around the world is carried out by counting the number of these animals, their mandatory routine immunization and evaluating the effectiveness of vaccination against the accumulation of rabies virus-neutralizing antibodies. Inactivated vaccines based on different viral strains formulated with a wide range of adjuvants are used to induce a protective level of humoral immunity against rabies in dogs (≥ 0.5 IU/cm3), which allows vaccines with high safety and effectiveness for the target animal species to be obtained. The article presents the results of the study of humoral immunity in 20 non-pedigree dogs 21 days after the administration of rabies inactivated vaccines based on culture rabies virus from ARRIAH strain formulated with various adjuvants. The presented rabies vaccines formulated with oil adjuvant Montanide ISA 70 VG and gel adjuvant Montanide GEL 01 were innocuous and safe and induced strong immunity in all vaccinated animals. The vaccine formulated with Montanide ISA 70 VG adjuvant in case of a single administration in the dose of 1.0 cm3 induces formation of rabies virus-neutralizing antibodies in the level of 2.4 times higher than the vaccine formulated with Montanide GEL 01 adjuvant. The highest levels of rabies antibodies in dogs were 48.1 ± 3.7 and 28.3 ± 1.5 IU/cm3 and were observed with the use of rabies inactivated emulsion vaccine in the doses of 3.0 and 1.0 cm3 respectively.

Rabies cases are still reported in various mammal species in the Russian Federation. Wild carnivores contacting with domestic and farm animals and transmitting the pathogen to them are the main source and natural reservoirs of rabies. The highest level of rabies incidence in livestock is observed among cattle. Thus, 158 new outbreaks were reported in the Russian Federation in 2017 where the virus was identifi ed in cattle. Rabies specifi c prophylaxis as well as assessment of its effi cacy based on the level of accumulated antirabies virus neutralizing antibodies currently occupy one of the leading positions in the modern system of veterinary and sanitary measures against rabies. The paper presents the study results confi rming effi cacy and safety of three inactivated rabies vaccines based on the “ARRIAH” strain and intended for rabies prevention in cattle. The adsorbed and two emulsion rabies vaccines, formulated with diff erent adjuvants demonstrated innocuity and safety, and induced post-vaccination immunity in all immunized animals at day 21 post inoculation. The mean titers of anti-rabies antibodies were as follows: for inactivated adsorbed vaccine – 4.02 ± 0.76 IU/cm3; for inactivated emulsion vaccines – 16.30 ± 2.03 and 20.73 ± 3.39 IU/cm3. As compared with the adsorbed vaccine, the emulsion vaccines formulated with Montanide ISA 206 and ISA 70 adjuvants induce stronger immunity.

The use of high quality nutrient media comprising protein hydrolysates as the main components for maintaining their nutrient value is a critical prerequisite for appropriate fnal product preparation in FMD vaccine biotechnology. The incoming control of raw materials, in particular testing blood protein hydrolysates of different batches for their quality, is of considerable importance. Currently, there are different methods for the determination of hydrolysate efcacy using cell cultures and unicellular organisms as test systems. We used suspended baby hamster kidney (ВНК-21) cell line as a model for testing blood protein hydrolysates in the raw materials intended for FMD vaccine production for their biological activity and toxicity. The validation of the proposed method was carried out to confrm the reliability of the test results obtained. The following main validation characteristics were determined through testing 560 blood protein hydrolysate samples: trueness, specifcity, sensitivity, as well as precision under repeatability and reproducibility conditions. The validation results for the proposed method demonstrated high reliability (Fisher’s criterion – 0.021; ≤0.050) and full compliance with acceptance criteria.

Procedure for validation of the method for indirect assessment of cultural FMD virus titre (T FMDV) in raw materials used for vaccine production with real time reverse transcription polymerase chain reaction (RT-PCRrt) based on determined amplifcation threshold cycle value (Ct) using linear regression model, TFMDV= –0.2956Ct+ 11.4650, is described in the paper. Testing results for 390 samples of cultural FMD virus were analyzed and basic validation characteristics of the proposed method: specifcity, accuracy, precision, limit of detection and limit of quantifcation, application scope and linearity, were defned. Validation results for the method for indirect assessment of FMD virus titre with RT-PCRrt met the acceptance criteria.

The research tasks covered the study of ВНК-21/2-17 cell DNA transformation dynamics during FMDV reproduction process. It was noted that the destruction of major cell population coincided with the increase in apoptotic cell number and detritus amount. Three hours post cell culture infection increase in apoptosis and detritus was observed, G 1-phase decreased by 17–21% and polynuclear cells grew by 2.3 times. In seven hours, the drastic rise in cell death was noted. It was established that at all stages of FMDV culture in ВНК-21/2-17 suspension cell line, diploid cells G1(2n) were predominant, being basic cells for the virus reproduction. Cells in synthetic (S) and G2and M-phases were less susceptible to virus. Using flow cytometry technique made it possible to quantify cell cycle phases during reproduction in FMDV cells. We also succeeded in comparing between these phases, virus livability and virus reproduction dynamics. The study of FMDV cytopathic effect in ВНК-21/2-17 cells demonstrated that one of the optimization trends in culture vaccine production include proliferation inhibitory factor use at a certain cell cycle phase.

The raw material for blood protein hydrolysate preparation is whole animal blood, its clots and other serum production wastes. The dependence of amino acid composition of blood protein hydrolysate on the season of the raw material preparation was studied. The research lasted three years. It was demonstrated that the amino acid composition changed depending on the season. The peak, as a rule, was during summer months when their amount increased by 1.2–2.3 times and during autumn and winter it went down by 1.2–1.4 times (the difference is considerable, р

Method for determination of foot-and-mouth disease (FMD) virus 146S component concentration with real-time reverse transcription – polymerase chain reaction (rtRT-PCR) in vaccine raw materials is developed. Negative regression model of С146S = (30.269 – Ct )/4.155 type allowing determination of FMDV 146S particle concentrations based on the amplification threshold cycle values (Ct ) is proposed. It has been experimentally proven that the quantity of the 146S component determined by the real-time RT-PCR method using developed regression model and contained in the inoculation dose of FMD vaccine confers protection to the vaccinated animals against generalized FMD of A, O, Asia-1 types. rtRT-PCR method is highly sensitive and allows rapid and highly reliable estimation of the 146S antigen concentration in FMD vaccine. The method for 146S particle quantity determination by real-time RT-PCR using the regression model is reliable and demonstrates high correlation (95.5–99.0%) with the complement fixation test results.

The raw material for blood protein hydrolysate preparation is whole animal blood, its clots and other serum production wastes. The dependence of ВНК-21/2-17 cell lineage productivity on the season of hydrolysate raw material preparation was observed. Cell growth stimulated by blood protein hydrolysate from raw material prepared in summer months 1.2–1.3 times exceeded cell growth stimulated by blood protein hydrolysate prepared in autumn-winter period (differences are significant, p < 0,001). The amount of FMDV immunogenic components from “summer” blood protein hydrolysate 1.3–1.6 exceeded the amount obtained during other seasons of the year (differences are significant, p < 0,001). There was found a direct correlation between BHK-21/2-17 cell lineage productivity, concentration of FMDV immunogenic components and seasonal dynamics of the contents of the following amino acids composing blood protein hydrolysates: alanin, asparagine, valine, histidine, glycine, isoleucine, leucine, lysine, methionine, proline, serine, tyrosine, threonine, tryptophane, and phenylalanine. To increase BHK-21/2-17 cell lineage productivity and concentration of FMDV immunogenic components it was suggested to add some additional amino acids into the culture growth medium for suspension cell culture in autumn-summer period together with blood protein hydrolysate

The paper presents the results of tests aimed to study the protective antigen differences between Asia-1 No. 2145/Tajikistan/2011 isolate and Asia-1 Shamir 3/89 FMD cultural virus vaccine strain.

The paper presents results of the ВНК-21/2-17 lineage cell cycle tests using flow cytometry. This cell line is widely used in biological industry that is why studying the ВНК-21/2-17 cell suspension pattern using the proposed technique and determination of tools and methods to prevent morphological and structural cell modifications in the culture affected by negative factors is of great interest. Flow cytometry allows to determine quantitative parameters of cell cycle phases and perform correlation between these phases and viability of the working cell population. It was mentioned that the general trend of this cell lineage suspension cultivation for 48 hours has a standard dynamics, herewith at all stages of the cell cycle the primary growth stage - G1 prevailed. Under unfavorable conditions of cultivation apoptotic debris could increase up to 31-59%, i. e. part of the cell population could be potentially non-reproductive. Unsuccessful cultivation stages associated with low pH content in the cultivator where pH was not maintained at the constant level were detected. During optimization of cell cultivation physical and chemical parameters proliferation intensity increased. Viable cell yield during cultivation when a specific pH was maintained was higher and at the synthetic stage G2 + M no cycle delay occurred.

The paper demonstrates results of the research aimed at optimization of amino acid composition of the nutrient media used for BHK-21-2/17 suspension cultivation by change of amino acid amount in such media. Growth level in the control and tested media amounted to 5.6; cell growth dynamics was also similar. In-media utilization of different amino acids was different. The cells mostly consumed glutamin, serine, tyrosine, methionin (up to 90%, 55%, 40% and 60%, respectively). Growth of alanine level was reported (from 10% in the control sample and up to 60% in the test sample). Amount of tryptophan and glutamic acid remained stable. Glycine, threonine, isoleucine were actively utilized within the first 24 hours (7-13%, 10-18%, 34-41%, respectively in the control experiment). Hereafter, the concentration slightly increased (by 7-45%, by 16-21%, by 20-50%, respectively in the control experiment). During the FMDV reproduction in the cells grown in the test and control media no significant differences in the yield of immunogenic virus components were reported.

The paper demonstrates the possibility to use white mineral oils resulting from hydrocatalytic processing of unconverted vacuum gas oil as a basis for an oil adjuvant used in the production of FMD vaccines intended for livestock. Samples of the developed white oils were tested for their reactogenicity and pyrogenicity, and emulsion vaccines were tested for their reactogenicity, potency, and emulsion stability. Tests results were compared with the ones for a foreign analogue. Blood samples were taken from animals before vaccination and 14 days after it to obtain sera which were further tested for FMD antibodies using VNT. Assessment of the white oil sample reactogenicity demonstrated that sample BM 1.4 obtained during hydroisomerization was the least reactogenic. Sample V-BM 1.4 demonstrated the best result in comparison with other samples within the group as well as in comparison with the control sample during the emulsion stability test. According to the potency test results, the emulsion vaccine based of the white oil sample BM 1.4 demonstrated the greatest difference in antibody titers in 14 days - 4.15 log2, while the one based on the control sample - 3.50 log2. This means that it the use of this vaccine results in 17.7-fold increase in FMD antibodies, while the use of control emulsion vaccine in 11.3-fold increase. Vaccines based on BM 1.3 and BM 2.2 oil samples demonstrated lower performance in comparison with the control vaccine in terms of these parameters.

The paper demonstrates results of FMD vaccine testing for their inability to induce antibodies to FMD virus non-structural proteins (FMDV NSP antibodies) in cattle at different dates post vaccination. Subtype A, O, Asia-1 and SAT-2 cultural FMD virus replicated in baby hamster kidney suspension culture (ВНК-21/2-17) was used for vaccine production. FMDV antigen was inactivated with aminoethyl ethylenimine solution, and the suspension was subsequently purified. Non NSP-purified preparation was made to simulate presence of NSPs in the vaccine. ELISA test-kits were used for testing cattle sera collected before and after immunization for presence of 3ABC polyprotein-specific antibodies. Inoculation of cattle with non-purified preparation induced antibodies specific to FMDV NSPs in 87.5% animals on day 30, in 75% animals on day 44 and in 62.5% animals on day 132 post third immunization. All tested vaccines manufactured by the FGBI “ARRIAH” were demonstrated to induce no FMDV NSP antibodies suggesting their complete removal from the virus-containing suspension.

The paper demonstrates dependence of virus-neutralizing antibody production and emulsion FMD vaccine immunogenicity on administration of vaccines against different infectious porcine diseases. Optimum interval between the administrations of the emulsion FMD vaccine and vaccines against other porcine diseases was determined.