RU2793900C1 - Method for indirectly determining the titer of the infectious activity of fmdv in non-inactivated raw materials for vaccines using a mathematical double differential of the crossing point data during amplification of viral cdna - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: production of foot-mouth disease vaccines (FMDVs), and in particular to a method for indirectly determining the titer of the infectious activity of foot-mouth disease vaccine in non-inactivated raw materials for vaccines using a mathematical double differential of the crossing point data during amplification of viral cDNA. The present invention combines the implementation of the neutralization reaction with the subsequent conduct of a molecular biological study, which increases the specificity and sensitivity of the method. It is proposed to use as a positive control of mRNA of the FMDV target site obtained after transient transfection of BHK-21/SUSP/ARRIAH cells with a gene construct obtained using the pJet 1.2 sf GFP plasmid vector. The dependence of the titer of infectious activity of FMDV and the values of crossing points for the graphs of the double mathematical differential, presented as lg regressive logarithmic function T_FMDV=-0.2997×C_p+11.675 was found, with high accuracy of approximation and efficiency of the amplification reaction.

EFFECT: within 3 hours and with a high degree of certainty, indirect determining the titer of the infectious activity of the cultured FMDV in non-inactivated raw materials for the vaccine based on the mathematical double differential of the crossing point data during amplification of the viral cDNA.

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Description

The invention relates to the field of biotechnology and the production of FMD vaccines, and in particular to a method for indirectly determining the titer of FMDV infectious activity in non-inactivated vaccine raw materials using a mathematical double differential of the crossing point data during amplification of viral cDNA.

Foot and mouth disease is a viral, most contagious, acute, febrile disease that affects wild and domestic artiodactyl and callus animals [1]. In accordance with the classification of the International Committee on Taxonomy, FMDV belongs to the order Picornavirales, the family Picornaviridae, the genus Aphthovirus, and the species food-and-mouth disease virus (FMDV) [2, 3]. To date, 7 serotypes of FMDV are known: O, A, C, Asia-1, SAT-1, SAT-2, SAT-3 with many topotypes and genetic lines [1]. For foot and mouth disease virus with a sedimentation constant of full viral particles 146 S, a diffusion constant of 1.41 cm ² /s, the molecular weight is 8.08 × 10 ⁶ D. The size of the virion is 23-25 nm, the mass is 8.4 × 10 ^-18 g [ 1, 4, 5].

The virus genome is represented by a single-stranded RNA molecule (ssRNA (+)) of positive polarity (Baltimore group IV of microorganisms). The virion RNA of different strains of foot and mouth disease virus have the same length of about 8400-8500 bp. with a molecular weight of 2.8 × 10 ⁸ MD [4]. Picornaviruses do not produce subgenomic mRNAs. RNA is simultaneously a template for genome replication and translation of viral proteins. The foot-and-mouth disease virus RNA comprises three distinct parts, namely a long 5'-untranslated region (5'-UTR) (about 1300 bp, which is rare in viruses) encoding a single open reading frame (ORF) region (in this is the similarity with the mRNA of eukaryotic cells) about 7000 n. and a 3'-untranslated region (3'-UTR) about 90 bp in length. (figure 1).

The VPg peptide is covalently linked to the 5' end of the genome and is encoded by the 3 B _1-3 genes. VPg exists in 3 different forms that play an important role in RNA synthesis. The 5'UTR region includes several different structural elements: S fragment, poly(C) tract (Cn), 3 or 4 pseudoknots (PK), cre/bus element, internal site for the ribosome (IRES) [5].

At the 5' end, there is a stable stem-loop region of 55 bp, which is necessary for transcript replication. This site was called the cis-acting replication element (cre) or 3B-site (bus)). It contains a conserved motif (AAACA) that acts as a template for uridylation of the VPg protein by viral RNA polymerase to form VPgpU and/or VPgpUpU. These products act as primers to initiate viral RNA synthesis, which explains the presence of VPg at the 5' end of RNA transcripts, both positive and negative sense [5, 6].

The translated region follows the 5'-UTR. The RNA is translated as a single long ORF into a polyprotein, followed by a series of post-translational proteolytic cleavages to form both intermediate and mature structural and non-structural proteins. This is the main part of the viral genome, which is about 7000 nucleotides long and includes 14 genes. It encodes a large polyprotein (about 2330 a.a.) that is rapidly cleaved by viral proteases to form four different structural and eleven non-structural proteins. After translation, four primary products are initially formed, namely Lpro (leader protease), P1-2A, P2, and P3 [7, 8].

The first component of the FMDV polyprotein is the L-protein (Leader), about 200 bp in size. The uniqueness of picornaviruses lies in the fact that a protease is used as a leader protein. Protein L is a papain-like cysteine protease. It cleaves autolytically from the rest of the viral polyprotein at the L/P1 junction. The L-coding region contains two separate AUG initiation codons (usually 84 nucleotides apart), which lead to the formation of two different L-proteases, named Lab and Lb, which differ from each other in length by 28 a.a. It is Lpro that is responsible for the inhibition of protein synthesis in the host cell by inducing cleavage of the host protein eIF4G. As a result, viral RNA can freely use the host cell's protein synthesis apparatus for its own protein synthesis [5, 8].

The P1-2A polypeptide (precursor of the capsid protein) is cleaved by the 3C protease (3Cpro) at the N-terminus and C-terminus with the help of the 2A protein to form proteins 1AB (VP ₀ ), 1C (VP ₃ ) and ID (VP ₁ ), 2A [ 9].

Proteins VP ₀ (1AB), VP ₃ (1C), and VP ₁ (ID) are structural components of natural empty capsids with a sedimentation coefficient of 75S. 60 copies of each of these peptides self-assemble, forming complete viral particles with a sedimentation coefficient of 146S [5, 8].

During the encapsidation of a region of the genome, the VP ₀ protein is cleaved to form the VP ₄ and VP ₂ proteins autolytically. The VP ₄ protein resides entirely within the viral particle, while VP ₁ , VP ₂ and VP ₃ reside on the surface and contribute to the antigenic properties of the virus. VP ₁ contains at least two important immunogenic sites, the GH loop (at amino acid positions 141-160) and the C-terminus (residues 200-213). The GH loop includes the arginine-glycine-aspartic acid (RGD) motif, which is required for virus attachment to the host cell via the integrin receptor [5, 9].

The VP ₁ protein is the most variable, since it accounts for more than 90% of all structural gene mutations. The most variable regions are regions 40–60, 130–160, and 190–213 a.a. The site of the surface protein VP ₁ in the region of 130-160 a.a. is characterized by high variability, since it is involved in the process of binding to host cell receptors [5]. The variability of this region allows FMDV to interact with receptors on different cell types and facilitates the transition from one host species to another. Nucleotide sequences of the VP ₁ coding region are used to genetically characterize FMD strains due to their importance for viral attachment and cell entry, protective immunity, and serotype specificity. Phylogenetic analysis based on the VP ₁ sequence is widely used to infer evolutionary dynamics, epidemiological relationships between genetic lineages, and to track the origin and movement of FMDV strains [8, 9].

Protein 2A is a very short peptide of 18 amino acid residues, which is a viral autoprotease. Apparently, this protein is responsible for the cleavage of the 2A/2B site, which is the second processing event in FMDV [5].

The P2 and P3 regions of the polyprotein are processed into FMDV non-structural proteins (NSPs). The P2 polypeptide undergoes cleavage into 2B proteins about 154 a.a. long. and 2C, about 318 a.a. using 3C protease. The function of protein 2B is not known. Protein 2C determines the resistance of the viral particle to guanidine. This protein is required for initiation of the negative RNA strand during synthesis [5, 9].

Protein P3 is cleaved by 3C protease to form 6 major proteins: 3A, three separate copies of VPg (3B ₁ , 3 B ₂ , 3 B ₃ ), 3Cpro and 3Dpol (3D-RNA polymerase), as well as intermediate products, in in particular, 3CD. Protein 3A is about 143 a.a. long. has hydrophobic sequences that presumably anchor it to membranes. Protein 3A can also serve to deliver 3B peptides to RNA replication sites [5, 7, 8].

The 3Cpro protease is 213 a.a. long. This enzyme belongs to the family of chymotrypsin-like serine proteases. It is responsible for most of the proteolytic processing of the viral polypeptide (10 cleavage sites), in particular, for the cleavage of P1-2A into VP ₀ , VP ₁ , VP ₃ proteins, as well as for the formation of various non-structural proteins. In addition, the 3C protein also modifies some cellular proteins. Thus, 3 proteases are required to cleave all VN proteins from the original polyprotein: the leading proteinase (L-protein), 2A, 3C [9].

The 3Dpol protein is encoded by a highly conserved 3D gene and has a size of 470 a.a. and is an RNA-dependent RNA polymerase that, together with other viral and possibly cellular proteins, replicates RNA from viral infectious RNA molecules. There are two processes required for viral RNA replication. Initially, the positive-sense genome is used as a template for antisense RNA synthesis, and this is then used to generate new infectious positive-sense RNA molecules [8, 9].

In a mature viral particle, there are 60 capsomeres, consisting of four structural proteins VP _1-4 , which bind to each other to form an icosahedral shell or capsid. The viral particle is used to deliver the genome of the infectious agent to the cytoplasm of the cell. Thus, the first step in viral replication is the translation of viral RNA to obtain each of the virally encoded proteins required for nucleic acid replication and for the production of viral particles capable of initiating a new cycle of infection [4, 5].

Proteins VP ₁ (1D), VP ₂ (1B) and VP ₃ (1C) are located on the surface of the virus and contribute to the antigenic properties of the virus, and VP ₄ (1A) is located inside the virion. On the surface, there is a GH-loop, which is characterized by hypervariability and includes 140-160 a.a. protein VP ₁ [5, 7].

The 3'UTR region is much shorter than the 5'UTR region, about 100 nucleotides in length. This site consists of a unique heterogeneous part with a size of about 90-100 b.p. and poly(A) moieties of about 50 n.o. The heterogeneous region folds to form a specific stem-loop structure followed by a variable length poly(A) fragment. The 3'UTR region plays an important role in viral genome replication. Interactions occur between the 5'-UTR and 3'-UTR, which can affect IRES activity and RNA replication [1,5].

In the process of reproduction in biological systems, the foot and mouth disease virus forms 4 variants of the components: 146S component (virion, complete particles), consisting of one whole molecule of viral RNA and 60 copies of the polypeptide, each of which is represented by a complex of proteins VP ₁ (1D gene), VP ₂ (1B gene), VP3 (1C gene), VP ₄ (1A gene); 75S particle ("empty" capsid), which includes 60 copies of the polypeptides VP ₀ (1AV-gene), VP ₁ (1D-gene), VP ₃ (1C-gene); 12S particle (capsomer) represented by structural proteins VP ₁ (1D-gene), VP ₂ (1B-gene), VP ₃ (1C-gene); 3,8S subunit represented by non-structural protein VP _g . The 75S, 12S, and 3.8S components do not include FMDV RNA [1,5].

Foot-and-mouth disease causes enormous economic damage, which is associated with a sharp decrease in the productivity of livestock, significant costs for the fight against the disease and the loss of trade opportunities. In terms of real production losses and vaccination costs in endemic regions alone, it is estimated that the cost of FMD to the economy is between US$6.5 billion and US$21.0 billion per year. Strengthening FMD control and vaccination of susceptible animals worldwide benefits both free and endemic countries and is seen as a global economic benefit [7, 10].

In the process of industrial production of FMD vaccines, special attention is paid to the titer of infectious activity of the virus before the inactivation of raw materials [2]. Based on this, raw materials for vaccines are examined for infectious activity with the determination of the titer using a monolayer transplantable porcine kidney cell line IB-RS-2 [3]. Significant disadvantages of this method are: 1) a long titration procedure associated with the development of a cytopathic effect (at least 3 days),

2) a certain degree of subjectivity in evaluating the results of the analysis,

3) the high cost of the cell line as a test system and the cost of maintaining it.

In this regard, it is advisable to search for a method for indirectly determining the titer of FMDV infectivity in non-inactivated vaccine raw materials using the mathematical double differential of the crossing point data during viral cDNA amplification.

This method makes it possible to reliably determine the titer of infectious activity of FMDV in raw materials for vaccines within 3 hours. Based on this, it is advisable to propose a new method for indirect determination of the FMDV titer in non-inactivated vaccine raw materials based on an alternative method.

The objective of the present invention is to develop a highly sensitive and highly specific express method for indirect determination of the titer of FMDV infectivity in non-inactivated vaccine raw materials in order to eliminate the above disadvantages.

This problem has been solved by developing a method for indirectly determining the titer of FMDV infectivity in non-inactivated vaccine raw materials using the mathematical double differential of the crossing point data during viral cDNA amplification. The proposed method allows:

1) to increase the specificity of sample analysis due to the reaction of neutralization of full particles of FMDV with strain-specific antibodies;

2) increase the sensitivity and specificity of the analysis through the use of highly specific original primers and a DNA probe designed for the 3D FMDV gene in the vaccine raw material;

3) control the real-time RT-PCR reaction using mRNA synthesized using the p Jet 1.2 sf GFP plasmid and BHK-21/SUSP/ARRIAH newborn Syrian hamster kidney cell culture [11] for transfection;

4) increase the reliability of the analysis by establishing the relationship between the titer of FMDV infectious activity (Tfmdv) and the point of the graph of double differentiation of the logistic crossing point curve (С _р ), presented as a logarithmic function: lg T _FMDV = -0.2997 × С _р + 11.675 with a high fit (R ² =0.9994) and an amplification efficiency of 99.46%. The proposed model will make it possible to indirectly determine the titer of infectious activity of FMDV in non-inactivated raw materials in the production of FMD vaccine.

The essence of the invention is reflected in the graphic images: Fig. 1 - Mapping of the FMDV genome.

Fig. 2 - The process of modifying the plasmid p Jet 1.2 sf GFP to clone the 3D FMDV gene region. The insertion is made between the T7 promoter and HDV ribozyme sites. The model is presented on DNA sections. Note: A - map of the plasmid with all sites, B - conditional map of the plasmid before insertion, C - the result of inserting the target region of the FMDV 3D gene into the plasmid, D - end sections of the plasmid and gene (left), E - end sections of the gene and plasmid (on right).

Fig. 3 - A model for constructing a primary logistic curve for a sample with an infectious activity titer corresponding to an amplification threshold cycle of 11.9 (blue), a plot of a mathematical differential (green), a plot of a double mathematical differential with a crossing point of 11.0 (red).

Fig. 4 - Dependence of the values of the crossing point of the logistic curves after mathematical differentiation on the titer of infectious activity of the FMDV in the raw material for vaccines (with indication of the standard error) (n=3, p<0.005).

Fig. 5 - Dependence of the titer of infectious activity of FMDV in raw materials for vaccines on the values of the crossing point of the logistic curves after mathematical differentiation from (with indication of the standard error) (n=3, p<0.005).

Fig. 6 - Design of original oligonucleotide primers and probe for indirect determination of FMDV infectivity titer in raw material for vaccines. Note: the reverse primer is given in two versions, namely, in the complementary direct and in the rev format - reverse complement.

The essence of the invention lies in a new approach for indirectly determining the titer of FMDV infectivity in non-inactivated raw materials for vaccines using the mathematical double differential of the crossing point data during amplification of the viral cDNA. The claimed method is based on carrying out a neutralization reaction of FMDV virions using purified polyclonal antibodies, obtaining a total RNA eluate, performing reverse transcription and real-time amplification reactions, conducting double mathematical differentiation of logistic curves for the studied samples of raw materials for the vaccine in the amplification reaction of FMDV cDNA .

Currently, a standard real-time amplification reaction is used to conduct a qualitative and quantitative study of FMDV suspensions, in particular, to determine the concentration of the 146S component and the titer of infectious activity [12, 13]. The literature describes a method for quantifying FMDV virions in non-inactivated vaccine raw materials by comparing the maximum extrema of the second derivative plots for real-time amplification reaction curves [14]. At the same time, this method was not used to determine the titer of infectious activity of FMDV in raw materials for vaccines.

Unlike the prototype, the developed method includes a neutralization reaction of FMDV virions using purified polyclonal antibodies from strain-specific blood sera of guinea pigs against FMDV; stage of sorption extraction of total RNA; stage of reverse transcription and amplification reactions using original oligonucleotide primers and probes; a new approach to the method of calculating the infectious activity titer of FMDV in raw materials for vaccines, taking into account the value of the crossing point for logistic curves subjected to double mathematical differentiation. The use of the developed method increases the reliability of the analysis to determine the titer of infectious activity of FMDV in samples of raw materials for vaccines. At the same time, as well as the prototype, the developed method allows you to simultaneously examine several dozen samples of non-inactivated raw materials for the vaccine, while the analysis time is reduced to 3 hours. Based on this, it is important to use this method to indirectly determine the titer of infectious activity of FMDV in the raw material for the vaccine.

The key element of the proposed method is the detection of crossing points for the logistic curves of the samples under study after double mathematical differentiation, on the basis of which the indirect determination of the titer of infectious activity of the FMD virus is carried out using the developed model of the relationship between the C _p point and the titer of the infectious activity of the FMD virus.

Comparative analysis with the prototype allows us to conclude that the novelty and inventive step of the claimed invention lies in the use of a method for indirectly determining the titer of FMDV infectivity in non-inactivated vaccine raw materials using a mathematical double differential of the crossing point data during amplification of viral cDNA.

Information on the development of the proposed method for indirect determination of the titer of infectious activity of FMDV in raw materials for vaccines was not found by the authors.

The essence of the invention is illustrated on the graphic material - graphs of the values of the crossing point of the logistic curves after mathematical differentiation and the titer of the infectious activity of the FMDV in the raw materials for vaccines relative to each other (figure 4, 5).

At the preparatory stage of the work, the immunological plate is sensitized with purified strain-specific polyclonal antibodies against foot-and-mouth disease virus in a volume of 2.00 cm ³ of a suspension with a concentration of immunoglobulins G of 5 μg/cm ³ . After immobilization of antibodies at a temperature of 4±2°C for 18-20 hours, the wells of the plate are washed three times with a standard TBST buffer solution, open binding sites are blocked with a 1.0% suspension of bovine serum albumin at a temperature of 37±0.5°C. for 30 minutes and again the wells are washed with standard buffer solution TBST 5 times. The procedure for preparing the plate for work with samples is carried out in advance, before the main analysis. Tablets can be prepared ahead of time and stored in a refrigerator at 4±2°C.

At the first stage of the study, the neutralization reaction of FMDV virions is carried out from the studied samples (dilutions of the standard, negative control, samples). A non-inactivated suspension of foot-and-mouth disease virus reproduced in a suspension culture of newborn Syrian hamster kidney cells (BHK-21/SUSP/ARRIAH) with a known concentration of viral particles is used as a standard. The negative control is a suspension of BHK-21/SUSP/ARRIAH cells not infected with viruses, bacteria, mycoplasmas and fungi at a concentration of 3.0-3.5 million cells/cm ³ . A control panel of ready-made dilutions of the standard is used with the content of viral RNA equivalent to the following titers of FMDV infectivity: 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 , 8.0, 9.0, 10.0 lg TCD ₅₀ /cm ³ . As part of the production process of greatest interest are suspensions with titers of infectious activity of the FMD virus above 6.0 lg TCD ₅₀ /cm ³ . In wells with sensitized strain-specific antibodies against FMDV make 2.0 cm ³ suspensions of samples and incubated at a temperature of 37±0.5°C for 30 minutes. As a result of a serological reaction, immune complexes are formed on the surface of the wells. The wells are washed of ballast components using standard TBST buffer solution 3 times. The resulting immune complexes are resuspended in 0.5 cm ³ 1/15 M phosphate buffer solution.

At the next stage of the analysis, RNA is isolated from the immune complex by the solid-phase sorption method. To 100 µl of immune complex suspension add 900 µl of 5M GTC, incubate the content during stirring for 5 minutes, then extend the incubation in an open dry thermostat at a temperature of 60 ± 2°C for 2 minutes. The resultant lysate is passed through a glass fiber column on a Promega Vac-Man Vacuum Manifold unit under negative pressure with a vacuum pump. At the next stage, fiberglass is washed from ballast components. To do this, 500 μl of a 40% solution of propanol-2 is added to each column with the vacuum pump running, and the liquid is expected to pass completely through the filter. To remove hydrophobic impurities, 500 ml of 80% propanol-2 solution are added to each tube, then the same procedure with 100% propanol-2 is added twice. After removal of the alcohol, 50 µl of the elution solution (TE standard buffer) are added to the columns and the contents are heated at a temperature of 60 ± 2°C for 8–10 min. After the desorption process, the columns are placed in eppendorf tubes with a volume of 1.5 cm ³ and centrifuged for 1.5 min at 13500 rpm on a table microcentrifuge, RNA eluates are taken. The resulting extract of total RNA is stored at a temperature of -20±2°C or immediately used in further work.

After obtaining the FMDV RNA extract, reverse transcription and amplification reaction are carried out using specific original oligonucleotide primers and a probe for the study of control samples and samples. To set up the reaction, a reaction mixture is prepared, the formulation of which is presented in Table 1. The following oligonucleotides are used as homologous to the 3D gene of foot-and-mouth disease virus:

(Fig.6) at a concentration of 5 pM per reaction. It should be noted that the oligonucleotide probe is direct relative to the reference nucleotide sequences of the cDNA nucleotides of all 7 types of FMDV. To form the nucleotide chains of the reaction products, deoxyribonucleoside triphosphates are used with a concentration of 0.3 mM each in the reaction mixture. As a basis, a PCR buffer of the following composition is used: 165 mM (NH ₄ ) ₂ SO ₄ , 675 mM Tris-HCl (pH 8.8), 0.1% Tween-20. 3 mM magnesium chloride, 3 mM manganese (II) chloride and dimethyl sulfoxide were added to the reaction mixture in an amount of 2% of the total volume without RNA eluate. Deionized water of the Milli Q purification system is used as a solvent. Tth (Thermus thermofilus) DNA polymerase (2 units) is used as a catalyst for reverse transcription and amplification reactions. This number of units made it possible to increase the efficiency of the amplification reaction without reducing the specificity of oligonucleotide annealing. Tth DNA polymerase is a thermostable 92-94 KDa protein isolated from a recombinant E. coli strain carrying the Thermus thermophilus KTP polymerase gene. Tth DNA polymerase catalyzes the polymerization of nucleotides in dsDNA in the direction from 5'→3' in the presence of Mg ²⁺ cations. Tth DNA polymerase also has reverse transcriptase activity in the presence of Mn ²⁺ cations. With the use of Tth DNA polymerase, it is possible to obtain amplifications from 100 to 8500 bp. Given that the size of the FMDV genome is 8500 bp, this served as an incentive to use this enzyme in a combined reverse transcription and amplification reaction.

The volume of the reaction mixture of components for one reaction is 20 μl. The RNA eluates of each sample are added to the mixture in 5 μl. The total volume of the mix is 25 μl.

The reaction is carried out at temperature and time parameters, information about which is presented in table 2. Reverse transcription is carried out at a temperature of 50°C for 20 minutes for 1 cycle, preliminary denaturation of complementary DNA - at a temperature of 95°C for 2 minutes for 1 cycle . The real-time amplification reaction is carried out for 45 cycles, each of which consists of 3 sub-stages: "denaturation" carried out at a temperature of 95 ° C for 20 s, as well as the sub-stages of "annealing of primers and probe" and "elongation and accumulation of fluorescent signal”, carried out at temperatures of 57°C for 20 s and 72°C for 20 s, respectively. The total process of thermal cycling takes 67 min, excluding transitions from one regime to another.

The results of the reaction are analyzed by evaluating and comparing the graphs of the accumulation of the fluorescent signal after double differentiation by the values of the crossing points (C _p ), determined by plotting the second derivative of the function F1=f (C _p ). The value of _Cp is an important characteristic of the reaction, it is directly proportional to the number of copies of the original viral RNA and, accordingly, the titer of the infectious activity of FMDV in the raw materials for vaccines. Taking into account that the function of the double differential f (С _р ) (f "(С _р )) is continuous in some neighborhood of the point С _р =C _p1 and is given on the interval of amplification cycles [0; 40], there is a certain area near the point С _р , for which in all coordinates on the axis 0-C _p the double mathematical differential of the function f (C _p ) will be negative. Since f "(C _p ) is the first differential of the function f '(C _p ), then from the condition (f '(C _р ))'<0, it follows that f'(С _Р ) on some small segment containing the point С _р =C _p1 , will be decreasing. Taking into account that f '(С _р )=0, in the section with С _р <C _p1 the first differential of the function f (С _р )>0, and with С _р >C _p1 we get that f '(Ср)<0. In other words, the first differential of the function f (С _р ) when passing through the point С _р =C _p1 changes its sign from “+” to “-”, therefore, at the point C _p1 , the function that reflects the process of accumulation of the fluorescent signal has the greatest value [15 ]. Thus, if the graph of the amplification reaction is represented by the function Fl1=f (C _p ), f '(C _p )=0 and f "(C _p )<0, then, provided that C _p =C _P1 , the resulting function has the greatest value at the point with the argument C _p1 , the value of which is taken into account to establish the relationship between the titer of infectious activity of FMDV in the raw material for vaccines and the value of the C _p function after double mathematical differentiation.

This method has an advantage due to the fact that when the curve is multiplied by any factors, the position of the largest values of the function does not change. The maximum on the graph of the double differential when it is examined for maxima is located inside the exponential section of the sigmoid, where the efficiency of the amplification reaction is a constant. The highest values of the first differential are, most often, in the zone of distortion of amplification efficiency values, so they are not recommended for analysis. Graphs of the third derivative give less accurate results, since their coordinates are largely associated with noise values [15].

Having calculated the _Cp values of the graphs of the double mathematical differential for curves reflecting the accumulation of the fluorescent signal of samples with different titers of FMDV infectious activity, the relationship between the virus titer in the non-inactivated raw material for the vaccine and the value of the _Cp point after double mathematical differentiation is established. Based on the developed model, the value of the titer of infectious activity of FMDV in non-inactivated raw materials for FMD vaccines is calculated.

Example 1. Obtaining a positive control for the indirect determination of the titer of infectious activity of the cultural foot and mouth disease virus in raw materials for vaccines.

In order to indirectly determine the infectious activity titer of the cultural FMDV in the raw materials for vaccines, the Golden Gate method of highly processive cloning was used, which makes it possible to create mRNA encoding the target region of the FMDV genome.

The pJet 1.2 sf GFP plasmid (size 3111 bp) with the green fluorescent protein gene (GFP) was used as an insertion vector for transfections into eukaryotic cells. A map of this plasmid is shown in Figure 2 A. This plasmid can function in bacterial cells and is resistant to ampicillin (AmpR). The pJet 1.2 sf GFP plasmid can be used to insert targeted gene regions between the T7 promotor and the HDV ribozyme. Hepatitis delta virus (HDV) ribozyme is a non-coding RNA found in hepatitis delta virus that is essential for virus replication and is the only known human virus that uses ribozyme activity to infect its host. This feature allows the synthesis of mRNA of the desired sequence, which can subsequently be used as a control for a quantitative amplification reaction, in particular, for indirect determination of the titer of FMDV infectivity in raw materials for vaccines.

Research was carried out on the construction of a modified section of FMDV RNA at positions 7843…7976 b.p. (section of RNA of the 3D gene of the virus:

или соответствующий участок ДНК:

or the corresponding section of DNA:

It is for the amplification of this RNA region that oligonucleotide primers and a probe were developed above.

Adapter sequences were "sewn" to the ends of the selected sequence using PNR:

('- сайты для разрезания рестриктазой Bsa I).

('- sites for cutting with restriction enzyme Bsa I).

These adapters were necessary for the formation of “sticky” ends formed after treatment with restriction enzyme II S. These enzymes introduce breaks not at the restriction site, but at some distance from it, in particular, for the Bsal restriction enzyme, the restriction and cutting sites are as follows: GGTCTCNNNNNN.

The adapter located at the 3'-end is placed with respect to reverse-complemente in the 5'→3' direction.

To obtain the modified sequence, PCR was performed using the following two primers (areas underlined for complementarity with plasmid sequences):

As a result, amplicons containing the target region and adapters were obtained:

The resulting end sections of the nucleotides in the target sequence and the plasmid are shown in Fig.2 B, 2 C, 2 D, 2 E.

At the next stage of the study, transient transfection of cells was performed. BHK-21/SUSP/ARRIAH cells were transiently transfected with high molecular weight polyethyleneimine (PEI) (60,000 kDa) (Acros Organics). The TurboFect transfection reagent (Fermentas) was used as a control in accordance with the manufacturer's instructions. The p Jet 1.2 sf GFP plasmid (Clontech) encoding the FMDV nucleic acid region was used as reporter genetic constructs. Two days before transfection, cells of the BHK-21/SUSP/ARRIAH line were seeded at a concentration of 0.45-0.5 × 10⁶ cells/cm³. Cells were transfected with a viability of more than 95% at a content of 1.5 - 2 × 10⁶ cells/cm³. Plasmid DNA and PEI were separately diluted in completely serum-free F12 medium at 1.0 and 2.0 µg/cm.³, respectively. PEI was added dropwise to the DNA solution. The mixture was stirred for 4 seconds and incubated at room temperature for 3 minutes. Then a mixture of DNA+PEI was added to the cells. Small scale transfections were performed on six well plates. Large scale transfections were performed in 2 liter shake flasks. After transfection, cells were harvested, resuspended in lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% polidocanol (detergent), 0.5% sodium deoxycholate).

Example 2. Revealing the existence of a relationship between the titer of FMDV infectivity in non-inactivated vaccine raw materials and the value of the crossing point after calculating the double mathematical differential of the primary logistic curves for samples during analysis in the cDNA amplification reaction.

At the first stage of the study, a panel of ready-made dilutions of the standard was used, which was used as a non-inactivated suspension of the cultural FMD virus with amounts of viral RNA equivalent to the following titers of infectious activity of the FMD virus: 0.0, 1.0, 2.0, 3.0, 4, 0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 lg TCC ₅₀ /cm ³ . In wells with sensitized strain-specific antibodies against foot and mouth disease virus, 2.0 cm ³ of sample suspensions were added and incubated at a temperature of 37±0.5°C for 30 minutes. As a result of the neutralization reaction, immune complexes were formed on the surface of the wells. The wells were washed from ballast components using standard TBST buffer solution 3 times. The resulting immune complexes were resuspended in 0.5 cm ³ 1/15 M phosphate buffer solution.

RNA was isolated from the immune complex by the solid-phase sorption method in accordance with the procedure presented above.

A reverse transcription and amplification reaction was carried out using specific original oligonucleotide primers and a probe for the study of control samples and samples. To set up the reaction, the reaction mixture was composed. The FMD-3D-Titer-F primer, FMD-3D-Titer-R primer, and FMD-3D-Titer-P-ROX/BHQ2 probe at a concentration of 5 pM per reaction were used as oligonucleotides homologous to the FMDV 3D gene. To form the nucleotide chains of the reaction products, deoxyribonucleoside triphosphates were used at a concentration of 0.3 mM each in the reaction mixture. The PCR buffer of the following composition was used as a base: 165 mM (NH ₄ ) ₂ SO ₄ , 675 mM Tris-HCl (pH 8.8), 0.1% Tween-20. 3 mM magnesium chloride, 3 mM manganese (II) chloride, and dimethyl sulfoxide were added to the reaction mixture in an amount of 2% of the total volume without RNA eluate. Deionized water of the Milli Q purification system was used as a solvent. Tth (Thermus thermofilus) DNA polymerase (2 units) was used as a catalyst for reverse transcription and amplification reactions. The volume of the reaction mixture of components for one reaction was 20 µl. The RNA eluates of each sample were added to the mixture in 5 μl. The total volume of the mix was 25 μl.

Reverse transcription was carried out at a temperature of 50°C for 20 min per 1 cycle, preliminary denaturation of complementary DNA - at a temperature of 95°C for 2 min for 1 cycle. The real-time amplification reaction was carried out for 45 cycles, each of which consisted of 3 sub-stages: "denaturation", carried out at a temperature of 95°C for 20 s, as well as the sub-stages of "annealing of primers and probe" and "elongation and signal”, carried out at temperatures of 57°C for 20 s and 72°C for 20 s, respectively. The total process of thermal cycling took 67 min, excluding transitions from one regime to another.

The results of the reaction were analyzed by evaluating and comparing the graphs of the accumulation of the fluorescent signal after double differentiation by the values of the crossing points (C _p ), determined by plotting the second derivative of the function F1=f (C _p ).

The data obtained were analyzed using the Rotor-Gene FRT-Manager software, which allows plotting the accumulation of a fluorescent signal in real time over a given number of amplification cycles (C). Using the technologies of the computer program "Maxima" (or equivalent), we plotted graphs after calculating the mathematical double differential for the obtained eluates of FMDV RNA of each dilution of the standard with certain values of the FMDV titer and calculated the average values of the crossing point with the projection onto the abscissa axis О-С ( n=3) (Fig. 3). The crossing point values for all dilutions of the FMDV standard with infectivity titers from 0.0 to 10.0 lg TCD ₅₀ /cm ³ ranged from 38.94±0.12 to 5.64±0.01, respectively. The standard deviation at a low titer of infectious activity (0.00 lg TCD ₅₀ /cm ³ ) is higher (0.12) than at a high titer (10.0 lg TCD ₅₀ /cm ³ ) due to a decrease in the sensitivity of the assay. In the study of the negative control, the accumulation of a fluorescent signal was not observed, which confirms the absence of foot-and-mouth disease virus in this sample. In the presented studies, the p-significance level is less than 0.005, which confirms the reliability of the quantitative studies. The dependence of the titer of infectious activity of the FMDV and the values of the crossing points for the graphs of the double mathematical differential is presented in Fig.5 and is reflected in the form of a regression logarithmic function lg T _FMDV = -0.2997 × C _p +11.675 with high accuracy of approximation R ² =0, 9994 and an amplification efficiency of 99.46%. The efficiency of the amplification reaction was calculated by analyzing the slope from the plot of the function C _p =-3.335×lg T _FMDV +38.95 with a high-fidelity approximation R ² =0.9994 presented in Fig.4. Thus, the existence of a dependence between the titer of FMDV infectious activity in non-inactivated raw materials for the vaccine and the crossing point for the graph constructed based on the function of the double mathematical differential of the primary logistic curves was revealed. The proposed model makes it possible to indirectly determine the infectious activity titer of FMDV in non-inactivated raw material for FMD vaccine.

Example 3 Indirect determination of FMDV infectivity titer using the mathematical double differential method of crossing point data in viral cDNA amplification.

Reference non-inactivated suspensions of cultured FMDV strains A/Turkey/2006, O/Saudi Arabia/2008, C/Zakarpatsky/1972, Asia-1/Tajikistan/2011, SAT-1/Akhalkalaki/1962, SAT-2 SAU7/ 2000, SAT-3 Bechuanaland 1/65 with varying infectivity titers as measured in the IB-RS-2 porcine kidney monolayer continuous cell line. As a positive control (K+) for quantitative analysis, a non-inactivated suspension of cultured FMD virus with an infectivity titer of 7.00 lg TCD ₅₀ /CM ³ was used. The positive control for qualitative analysis of reverse transcription and amplification reactions was messenger RNA obtained using the plasmid system obtained in example 1. cells/cm ³ . Test samples and control samples were tested in triplicate. All stages of the analysis were carried out as presented in example 2.

The results of the study are presented in table 3, from which it follows that C _{p average K+} was 15.61±0.01, which corresponded to a titer of 7.00±0.01 lg TCD ₅₀ /cm ³ (coincides with the reference value). C _p for samples of strains A/Turkey/2006, O/Saudi Arabia/2008, C/Zakarpatsky/1972, Asia-1/Tajikistan/2011, SAT-1/Akhalkalaki/1962, SAT-2 SAU7/2000, SAT-3 Bechuanaland 1/65 were 13.87±0.04, 10.77±0.03, 11.44±0.04, 8.13±0.03, 15.67±0.02, 12.17±0 .01, 12.94±0.03, respectively. Using the developed model:

determined the values of the titer of infectious activity of the foot-and-mouth disease virus, which were respectively equal to 7.52±0.04, 8.45±0.03, 8.25±0.04, 9.24±0.03, 6.98±0, 02, 8.03±0.01, 7.80±0.03, 7.00±0.01 lg TCD ₅₀ /cm ³ , which was combined with the analysis data in the monolayer transplantable pig kidney cell line IB-RS-2. In the negative control, FMDV was not detected by any of the indicated methods. The analysis took no more than 3 hours. Thus, the developed method allows you to quickly indirectly determine the titer of FMDV infectivity in non-inactivated vaccine raw materials using the mathematical double differential of the crossing point data during viral DNA amplification.

Example 4. Determination of the degree of reliability of the indirect determination of the titer of FMDV infectivity in raw materials for vaccines using the method of mathematical double differential of the data of the crossing point in the amplification of viral cDNA.

For the analysis used 460 samples of the suspension of the cultural foot-and-mouth disease virus with titers of infectious activity of 1.00-10.0 lg TCD ₅₀ /cm ³ . Positive and negative controls were the same as in examples 2, 3. Quantitative analysis was carried out as reflected in example 2.

With the obtained extracts of FMDV RNA, reverse transcription and cDNA amplification reaction were performed, followed by detection of analysis products using the developed original specific oligonucleotide primers and a probe, calculations were performed using a logarithmic regression function: lg Tfmdv = -0.2997 × С _р +11.675.

The presented samples and controls were also tested in a monolayer transplantable porcine kidney cell line IB-RS-2. The results of the analysis are presented in Table 4. It was found that the data obtained using the developed method correlated with the classical method by 98.5-100.0% for virus titers of 7.0-10.0 lg TCD ₅₀ /cm ³ (n= 115), by 97.1-98.4% for 6.00-6.99 lg TCD ₅₀ / cm ³ (n=115), by 95.9-97.0% for 2.00-5.99 lg TCD ₅₀ /cm ³ (n=115), by 94.3-95.8% for 1.00-1.99 lg TCD ₅₀ /cm ³ (n=115).

In other words, the developed method makes it possible to quickly and reliably determine the infectious activity titer of the cultured FMDV in non-inactivated raw materials for vaccines.

Example 5. Determination of diagnostic indicators of the developed method

To study the presented method (the proposed invention) for indirect determination of the titer of infectious activity of FMDV in raw materials for vaccines, standard diagnostic indicators were studied [16, 17]. To determine the sensitivity of the developed method, 450 samples of virus-containing non-inactivated raw materials were analyzed, which were known to be positive according to the analysis data in a sensitive transplantable monolayer transplantable porcine kidney cell line IB-RS-2. The titer of the virus for the test samples was in the range of 6.0-10.0 lg TCC ₅₀ /cm ³ . The formulation of the reaction and the differential mathematical analysis were carried out as shown in example 2. Using the developed method (the proposed invention), it was determined that out of 450 sample samples, 450 were identified as positive, 0 as negative. To study the specificity of the method, 140 negative samples that did not contain FMDV were tested. As a result of the study using the developed method (the proposed invention), it was determined that out of 140 negative samples - 140 were identified as negative. Using the above statistical methods of analysis, it was determined that the diagnostic sensitivity (DSe) was 100% (in the 95% confidence interval: 99.18-100.0%), the diagnostic specificity (DSp) was 100% (in the 95% confidence interval: 98.08-100.0%), k-test - 1.000; positive predictive value (PPV) - 100%, negative predictive value (NPV) - 100%, overall accuracy (DAc) - 100% (95% confidence interval: 99.43-100.0%) (Table 5) .

The main advantage of the present invention is the possibility of indirect determination of the infectious activity titer of the cultured FMDV in non-inactivated raw materials for vaccines. The present invention combines the formulation of the neutralization reaction with the subsequent molecular biological study of a non-inactivated suspension of the cultural FMD virus, which increases the specificity and sensitivity of the method. The present invention proposes the use as a positive control of the mRNA of the target region of foot-and-mouth disease virus obtained by expressing the plasmid modified pJet 1.2 sf GFP vector in BHK-21/SUSP/ARRIAH cells after transient transfection. The present invention uses Tth DNA polymerase, which makes it possible to combine the steps of reverse transcription and amplification reactions, as well as original specific oligonucleotide primers and a probe calculated for the target region of the 3D gene of foot-and-mouth disease virus of different industrial strains, which makes the method used universal. In the present invention, the dependence of the titer of FMDV infectious activity and the values of the crossing points for the graphs of the double mathematical differential is presented as a regression logarithmic function lg T _FMDV - -0.2997 × C _p +11.675 with a high approximation reliability of 0.9994 and an amplification efficiency of 99, 46%. The developed model allows for the indirect determination of the infectious activity titer of the cultural FMDV in the raw materials for the manufacture of vaccines.

The present invention makes it possible to quickly (in 3 hours) indirectly determine the infectious activity titer of the cultured FMDV in non-inactivated vaccine raw material based on the mathematical double differential of the crossing point data during viral cDNA amplification.

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 "Method for indirect determination of the titer of FMDV infectivity in non-inactivated raw materials for the vaccine using the mathematical double differential of the data of the crossing point during amplification of viral cDNA":

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11. Patent of the Russian Federation No. 2 722 671, 01.10.2019. BHK-21 / SUSP / ARRIAH - transplantable suspension subline of kidney cells of a newborn Syrian hamster, intended for the reproduction of foot-and-mouth disease, rabies, parainfluenza-3, Aujeszky's disease viruses in the production of antiviral vaccines, as well as for the manufacture of diagnostic and prophylactic veterinary biological products // Application No. 2019131190 . 01.10.2019 / Lozovoi D.A., Guseva M.N., Mikhalishin D.V. [and etc.].

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Claims (11)

1. A method for indirectly determining the titer of FMDV infectious activity in non-inactivated vaccine raw materials using the mathematical double differential of the crossing point data during viral cDNA amplification, which includes the following steps: - the reaction of strain-specific neutralization of foot-and-mouth disease virus in raw materials for vaccines; - extraction of total RNA from immune complexes after the neutralization reaction using the solid-phase sorption method; - carrying out reverse transcription and amplification reaction with detection of the fluorescent signal of the logistic curves of the studied samples using the developed original oligonucleotide:

and molecular probe FMD-30-Тiter-Р(ROХ/ВНQ2)-probe with design

which are designed for the target region of the FMDV 3D gene; - calculation of the crossing point using the function of the double mathematical differential of logistic curves; - determination of the infectious activity titer of the cultural foot-and-mouth disease virus in samples of non-inactivated raw materials for the vaccine based on the developed logarithmic regression function of the dependence of the virus titer and the crossing point lg T _FMDV = -0.2997×C _p +11.675 with an approximation reliability of 0.9994 and an amplification efficiency of 99 .46%.. 2. The method according to claim 1, characterized in that the enzyme Tth DNA polymerase is used in the amount of 2 units of activity in complex with manganese (II) chloride at a concentration of 3 mM per 1 reaction to combine reverse transcription and amplification reactions in time and space. 3. The method according to claim 1, characterized in that mRNA of the target region of the FMDV 3D gene obtained as a result of transient transfection of a suspension continuous cell line of the kidney of a newborn Syrian hamster BHK-21 / SUSP / ARRIAH is used as a positive control with a gene construct obtained using a modified pJet 1.2 sf GFP plasmid vector constructed using Golden Gate technology. 4. The method according to p. 1, characterized in that the analysis time is reduced to 3 hours and the following indicators of diagnostic sensitivity, specificity, accuracy and k-criterion are achieved in a 95% confidence interval: 98.5-100%, 98, 08-100.0%, 99.43-100.0% and 1.000 respectively.

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