INTRODUCTION. The completeness of virus inactivation and the identity of the vaccine strain are essential parameters for the safety and quality of inactivated virus vaccines, which should be controlled during vaccine development and production. Currently, the most promising quality control methods for inactivated virus vaccines are molecular genetic methods, which provide rapid results with high sensitivity and specificity.AIM. The aim of this study was the development of a real-time quantitative polymerase chain reaction (qPCR) method and an integrated cell culture real-time quantitative polymerase chain reaction (ICC-qPCR) method to assess the completeness of virus inactivation, as well as a reverse-transcription polymerase chain reaction assay coupled with restriction fragment length polymorphism analysis (RT-PCR-RFLP) to confirm the identity of the vaccine virus strain.MATERIALS AND METHODS. This study used RNA of CHIKV genotypes (three strains of each of the four CHIKV genotypes, including Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL), which were identified by sequencing prior to analysis). Additionally, the study used the Nika21 CHIKV strain (ECSA genotype), the Nika21 CHIKV strain inactivated with β-propiolactone, and the Nika21 CHIKV strain antigen adsorbed on aluminium hydroxide. The methods used included real-time qPCR, RT-PCR-RFLP, and virus neutralisation.RESULTS. The study identified a 218 bp fragment of the nsP1 gene (positions 789 to 1006) with restriction endonuclease recognition sites. These sites were present or absent in combinations specific to each of the four CHIKV genotypes. The authors selected primers for amplification of the specified gene region and tested the conditions for real-time qPCR and RT-PCR-RFLP. The study demonstrated the possibility of using the ICC-qPCR method to confirm the completeness of virus inactivation and the RT-PCR-RFLP method to identify the vaccine strain.CONCLUSIONS. The study showed the advantages of using the ICC-qPCR method to confirm the completeness of antigen inactivation and the RT-PCR-RFLP method to identify the vaccine strain. These methods are more sensitive and faster than traditional culture methods. ICC-qPCR and RT-PCR-RFLP can be used at any stage of the production process for inactivated vaccines.

Influenza A viruses (IAV) are a high threat to humanity because of a lack of proper effective antiviral drugs and resistance of viruses to existing vaccines. We describe the sufficient anti-IAV effect of Ans/PL-Dz nanocomposites that contain deoxyribozymes (Dz) immobilized on anatase TiO2 nanoparticles (Ans) through polylysine linker (PL). The Dz-containing nanocomposites appear to be more efficient than the Ans/PL-ODN nanocomposites that contain common oligodeoxyribonucleotides (ODN) targeted to the same RNA regions of the viral genome. The simultaneous use of nanocomposites that contain Dz and ODN, which are targeted to different sites of viral RNA provides a higher overall effect than the independent action of each of them (synergism). The inhibition of IAV with the proposed nanocomposites was shown to be effective, sequence-specific, and dose-dependent. The most efficient Ans/PL-Dz nanocomposite exhibited a high antiviral effect in vivo on mice models. The efficiency of IAV inhibition with this nanocomposite in vitro and in vivo is higher than that for the approved antiflu drug oseltamivir. The results open the prospect of creating a unique antiviral agent suitable for IAV suppression.

INTRODUCTION. Chikungunya virus (CHIKV) genotyping involves sequencing fractions of genes encoding E1, E2, nsP1 proteins or the entire genome of the virus. Available reagent kits or polymerase chain reaction protocols cannot be used for CHIKV genotyping, and nucleic acid sequencing requires expensive equipment and materials, which are not always available. Therefore, it seems promising to use a simpler and more cost-effective restriction fragment length polymorphism (RFLP) method, which has not previously been used for CHIKV genotyping.AIM. This study aimed to investigate the possibility of using reverse transcription polymerase chain reaction (RT-PCR) and RFLP for CHIKV identification and genotyping.MATERIALS AND METHODS. The experimental study used RNA from CHIKV strains of four genotypes, including the Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL). The study used RT-PCR followed by DNA restriction and restriction fragment length analysis.RESULTS. The nsP2 gene fragment of 648 bp in length (positions 3806 to 4453) contains recognition sites for the restriction endonucleases PspEI, PvuII, and DraI. The presence or absence of these sites generates a different combination specific to each of the four CHIKV genotypes. The authors designed primers for amplification of the selected gene region and performed RT-PCR and RFLP.CONCLUSIONS. The RFLP method can be used for rapid CHIKV identification and genotyping. The method provides results within a few hours and does not require high-tech equipment.

Single nucleotide polymorphism (SNP) is a change of one nucleotide by another. This change often leads to an emergence (or disappearance) of a site recognized by a certain restriction endonuclease. As a result amplification of DNA fragment using primers surrounding SNP point (containing either N1 or N2 nucleotide) followed by hydrolysis of the amplicon with this restriction enzyme will be different for three possible variants in a diploid genome (genotypes N1/N1, N1/N2 and N2/N2). This method of restriction fragments length polymorphism (RFLP) is widely used in the genetic studies. Earlier we have developed GlaI- and FatI-PCR analyses methods which allowed to carry out real-time PCR and showed it applicability for SNP determination. In the current work a new way to determine the single nucleotide polymorphism G/C by the Bst2UI-PCR analysis is proposed. GlaI- and Bst2UI-PCR analyses have been used to determine the frequency of G/C polymorphism variants at the chr20:37352001 position (according to GRCH38.p14 genomic assembly) in the blood DNA samples of 161 donors. The study included: 1) the isolation of leukocyte DNA from blood cells; 2) GlaI- and Bst2UI-PCR analyses of the DNA fragment chr20:37351957-37352083, 3) determination of cytosine and guanine at the chr20:37352001 position in the analyzed DNA preparations, and 4) comparative analysis of the obtained results. It has been shown that 68 donors (42.2%) have a heterozygous set of G/C at the chr20:37352001 position, 89 donors (55.3%) are homozygous by G, and 4 donors (2.5%) are homozygous by C. Thus, taking into account that blood cells have a diploid set of chromosomes, G to C replacement occurs in 76 out of 322 analyzed cases (23.6%). At the same time, from the results obtained it follows that the cytosine residue complementary to G at the chr20:37352001 position exists in methylated form (5-methylcytosine) in most of the DNA molecules, both in homo- and heterozygotes. The proposed method of Bst2UI-PCR analyses extends the possibilites of SNP determination using real-time PCR.

Rabies is an acute viral disease caused by a virus of the Rhabdoviridae family of the Lyssavirus genus, which affects the central nervous system and is characterised by absolute mortality. Vaccination is the only way to prevent the disease in humans. One of the products used for vaccination is a cultural concentrated purified inactivated dry rabies vaccine produced by the Federal State Budgetary Institution of Science “Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences” (hereinafter—Chumakov Center).The aim of the study was to examine the structure of the working virus seed of Vnukovo-32 strain used by the Chumakov Center for rabies vaccine production, to assess its genetic stability during production, to explore the possibility of using molecular genetic methods for identification of the production strain in the finished dosage form, and to study the nucleotide sequence of the CVS strain.Materials and methods: Vnukovo-32 rabies virus production strain, working virus seeds, finished batches of the rabies vaccine, CVS fixed rabies virus strain used in the assessment of specific immunity. The molecular genetic study was performed using RT-PCR followed by restriction and sequencing.Results: the paper presents the results of nucleotide sequence analysis of the G gene fragment obtained from the Vnukovo-32 production strain, batches of the working virus seed, and finished batches of the rabies vaccine produced in 2012, 2018, and 2019, and the CVS fixed rabies virus strain used in the assessment of the vaccine’s specific immunity. The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.Conclusion: Vnukovo-32 and CVS strains used by the Chumakov Center are rabies viruses. Analysis of the nucleotide sequence of the G gene fragment showed that the Vnukovo-32 strain remains stable throughout different production stages. The obtained nucleotide sequence of gene G of the Vnukovo-32 strain was deposited in GenBank (accession number MN116503). The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.

At early stages of carcinogenesis, the regulatory regions of some tumor suppressor genes become aberrantly methylated at RCGY sites, which are substrates of DNA methyltransferase Dnmt3. Identification of aberrantly methylated sites in tumor DNA is considered to be the first step in the development of epigenetic PCR test systems for early diagnosis of cancer. Recently, we have developed a GLAD-PCR assay, a method for detecting the R(5mC)GY site in the genome position of interest even at significant excess of DNA molecules with a non-methylated RCGY site in this location. The aim of the present work is to use the GLAD-PCR assay to detect the aberrantly methylated R(5mC)GY sites in the regulatory regions of tumor suppressor genes (brinp1, bves, cacna2d3, cdh11, cpeb1, epha7, fgf2, galr1, gata4, hopx, hs3st2, irx1, lrrc3b, pcdh10, rprm, runx3, sfrp2, sox17, tcf21, tfpi2, wnt5a, zfp82, and znf331) in DNA samples obtained from gastric cancer (GC) tissues. The study of the DNA samples derived from 29 tumor and 25 normal gastric tissue samples demonstrated a high diagnostic potential of the selected RCGY sites in the regulatory regions of the irx1, cacna2d3, and epha7 genes; the total indices of sensitivity and specificity for GC detection being 96.6% and 100%, respectively.

Aim. To study the genetic stability of the measles virus strain Leningrad-16 (L-16) used for the production of vaccine at JSC NPO Mikrogen.Materials and methods. A series of production and sowing strains of L-16 (JSC NPO Mikrogen), ready-made series of measles vaccines from various manufacturers, and the strain of measles virus genotype D6 were studied. Molecular genetic study of the strains was performed using RT-PCR followed by restriction analysis and sequencing.Results. The complete genome sequences of the production and sowing strains of L-16 that are used for vaccine production were obtained. The sequence of the vaccine strain was deposited in GenBank. Strain L-16 was confirmed to be genetically stable. The obtained data demonstrated the possibility of using the RT-PCR method with subsequent restriction analysis to confirm the authenticity of the vaccine strain L-16 in finished mono and three component vaccines.Conclusion. The results of the study suggest the applicability of the molecular genetic methods to confirm the authenticity of the studied strains not only at the production stages, but also in the finished series of vaccines.

Hypermethylation of the gene regulatory regions are common for many cancer diseases. In this work we applied GLAD-PCR assay for identificating of the aberrantly methylated RCGY sites in the regulatory regions of some downregulated genes in tissue samples of lung cancer (LC). This list includes EFEMP1, EPHA5, HOXA5, HOXA9, LHX1, MYF6, NID2, OTX1, PAX9, RARB, RASSF1A, RXRG, SIX6, SKOR1 and TERT genes. The results of DNA samples from 40 cancer and 25 normal lung tissues showed a good diagnostic potential of selected RCGY sites in regulatory regions of MYF6, SIX6, RXRG, LHX1, RASSF1A and TERT genes with relatively high sensitivity (80.0 %) and specificity (88.0 %) of LC detection in tumor DNA.

Aim.In order to study rubella virus strain RA-27/3 genetic stability, used for the vaccines production, a molecular genetic study was conducted.Materials and methods.In the study different series of master and work seed of RA-27/3 rubella virus strain by «Microgen», a few lots of rubella vaccines by the different manufacturers, as well as strain «Orlov» of rubella virus were used. RT-PCR followed by restriction, sequencing were performed .Results.Full-genomic sequences of the rubella virus strain RA-27/3 by «Microgen», were obtained and presented to GenBank. The full structure correspondence of RA-27/3 rubella virus strain by «Microgen» to the similar rubella strains used by GSK and Merck & Co Inc. has been shown. The RT-PCR method with the subsequent restriction was fulfilled using only domestic reagents. The developed method has been demonstrated as applicable for the identification of the RA-27/3 rubella virus strain as in monopreparation as well as in the combined vaccine preparation.Conclusion.The data obtained make it possible to suggest application of the molecular genetic methods for the vaccine virus identification not only at the production stages, but also in the finished vaccine lots.