[Adaptation of african swine fever virus (Asfarviridae: Asfivirus)to growth in the continuous culture PPK-66b cells by the method of accelerated passaging].

INTRODUCTION: African swine fever virus (ASF) is a large, enveloped virus with an icosahedral capsid morphology and a double-stranded DNA genome ranging in size from 170 to 190 kb. The replication cycle proceeds in two phases, the early phase lasting 4-6 hours and the late 8-20 hours after infection. The adaptation of the ASF virus to growth in continuous cell lines makes efficient and reliable genetic analysis and more accurate interpretation of its results. OBJECTIVE: Adaptation of a new isolate of the ASF virus to growth in a continuous cell line by the method of accelerated passages and preliminary genetic analysis of the resulting strain. MATERIALS AND METHODS: For virus isolation and passaging of the ASF virus, a porcine leukocyte cell culture (PL) and continuous cell cultures of porcine origin (ST, PK, PPK-66b) were used with Eagle MEM and HLA essential media with 10% porcine or fetal serum. RESULTS: The article presents data on the isolation and analysis of the changes in the reproductive properties of a new African swine fever (ASF) virus isolate in the process of adaptation to growth in a continuous piglet kidney cell culture clone b (PPK-66b). The current state of the problem of cultivation of the ASF virus, the features of its reproduction, and the basis of the genetic differentiation of its isolates are described in detail. Understanding the uniqueness of the nature of the ASF virus determined the approaches to the processes of its cultivation and adaptation. In this regard, the results of studies of cultural properties, and analysis of the nucleotide sequence of 6 genes of the new isolate, as well as phylogenetic analysis of these genes with already known strains and isolates of the ASF virus are presented. CONCLUSION: A new strain obtained in the process of cell adaptation of ASVF/Znaury/PPK-23 ASF virus by the accelerated passaging method reaches a high level of reproduction in 72 hours with an accumulation titer of 7.07 lg HAdE50/cm3. Primary genetic analysis allowed to establish the main phylogenetic relationships of the newly isolated strain with previously known variants of the current ASF panzootic.

[Alternative approaches to the diagnosis of African swine fever in the Russian Federation in 2017-2021].

INTRODUCTION: Prevention and control of African swine fever (ASF) transmission on the territory of the Russian Federation requires monitoring based on testing of samples from pigs and wild boars. Specific anti-ASFV antibodies are rarely detected in samples during routine serological diagnostics. Although, ASF isolates with weakened virulence were confirmed in Russia and neighboring countries.The aim of this work was to determine the possibility of using alternative samples for ASF diagnosis and evaluate the effectiveness of the diagnostic methods used on the territory of Russia. MATERIALS AND METHODS: Biological materials obtained from experimentally infected animals and samples collected in the "field" conditions were used in this study. RESULTS: Complex testing (RT-PCR and ELISA) is a more effective approach to diagnose chronic and asymptomatic forms of ASF compared to the separate use of these techniques. The possibility and efficiency of using alternative samples in diagnostics are demonstrated. It was confirmed that IPT method overcomes ELISA by high diagnostic sensitivity and detection of antibodies on earlier stages in extended range of samples. Anti-ASFV antibodies were detected in domestic and wild pigs in five regions of Russia. Samples from infected pigs that are negative in RT-PCR can be positive for anti-ASFV antibodies. The detection of antibodies in samples from shot wild boars (negative or uncertain in RT-PCR test) suggests the existence of animals surviving ASF infection. CONCLUSION: The data obtained suggest a revision of the ASF surveillance strategy, by introducing complex diagnostic methods aimed at detection of both the virus genome and anti-ASFV antibodies simultaneously.

[Problems of specific prevention of African swine fever].

This review presents the current state of the problem of development and application of the specific prevention of African swine fever (ASF) with a brief description of its etiology and pathogenesis. The unique nature of the ASF virus (ASFV) determines some limitations and the complexity of solving the problem of vaccine development. Such situation stimulated the development of highly specific diagnostic methods for rapid and accurate detection of the ASFV. In this regard, results of studies, including our own, concerning the comparative analysis of the genome of vaccine and virulent strains of the ASFV, as well as immunodiagnostic approaches to determine causes of high virulence and low protective activity of the ASFV, are briefly presented. Special attention is given to the issue related to the development of safe and effective vaccines against ASF. In this context disadvantages and possible advantages of live attenuated (LAV) and recombinant (RV) vaccines are considered in details. Results of recent studies on the assessment of the immunogenicity of genetically modified vaccines (GMV) which developed in various laboratories around the world are presented. The obtained data indicate that ASF vaccination is currently the most promising measure to stop the spread of this disease in our country and in the world, however, previous experience with ASF vaccination has revealed some problems in its development and application. The significant contribution of foreign researchers to the study of the basics of virulence of this pathogen and the study of its genes functions are noted. The possible further expansion of ASF in Europe and Asia in bordering Russia territories, as well as the established fact of the persistence of ASFV in wild boar population indicate a constant threat of its re-introduction into our country. In conclusion, the importance of developing a safe effective vaccine against ASF and the assessing of the possible risks of creating the artificial sources of the infection in nature as a result of its use is emphasized.

[Analysis of the whole-genome sequence of an ASF virus (Asfarviridae: Asfivirus: African swine fever virus) isolated from a wild boar (Sus scrofa) at the border between Russian Federation and Mongolia].

INTRODUCTION: The causative agent of African swine fever (Asfarviridae: Asfivirus: African swine fever virus) (ASF) is a double-stranded DNA virus of 175-215 nm. To date, 24 of its genotypes are known. Clustering of ASF genotype II isolates is carried out by examining a limited number of selected genome markers. Despite the relatively high rate of mutations in the genome of this infectious agent compared to other DNA viruses, the number of known genome molecular markers for genotype II isolates is still insufficient for detailed subclustering. The aims of this work were the comparative analysis of ASFV/Zabaykali/WB-5314/2020 virus isolate and determination of additional molecular markers which can be used for clustering of viral genotype II sequences. MATERIAL AND METHODS: ASF virus isolate ASFV/Zabaykali/WB-5314/2020 was used to extract genomic DNA (gDNA). Sequencing libraries were constructed using the Nextera XT DNA library prepare kit (Illumina, USA) using the methodology of the next generation sequencing (NGS). RESULTS: The genome length was 189,380 bp, and the number of open reading frames (ORFs) was 189. In comparison with the genome of reference isolate Georgia 2007/1, 33 single nucleotide polymorphisms (SNPs) were identified, of which 13 were localized in the intergenic region, 10 resulted to the changes in the amino acid sequences of the encoded proteins, and 10 affected the ORF of ASF virus genes. DISCUSSION: When analyzing intergenic regions, the ASFV/Zabaykali/WB-5314/2020 isolate is grouped separately from a number of isolates from Poland and three isolates from People's Republic of China (PRC), since it does not harbor additional tandem repeat sequence (TRS). At the same time, the construction of a phylogenetic tree based on DP60R gene sequencing relates ASFV/Zabaykali/WB-5314/2020 to isolates from PRC and Poland. Moreover, phylogenetic analysis of full-genome sequences confirmed previous studies on the grouping of viruses of genotype II, and as for the studied isolate, it was grouped with the variants from China. CONCLUSION: A new variable region was identified, the DP60R gene, clustering for which gave a result similar to the analysis of full-length genomes. Probably, further study of the distribution of ASF virus isolates by groups based on the analysis of this gene sequences will reveal its significance for studying the evolution of the virus and its spread.

[Changes in the genome of African swine fever virus (Asfarviridae: Asfivirus: African swine fever virus) associated with adaptation to reproduction in continuous cell culture].

INTRODUCTION: African swine fever virus (ASFV) is a large, double-stranded DNA virus in the Asfarviridae family. It is the causative agent of African swine fever (ASF). Only the genome of BA71V strain, adapted to Vero cell culture, was fully analyzed.The aim of this study was analyzing the complete genome sequence of two strains of adapted to the growth in CV-1 cell culture (CC) ASFV obtained after 30 and 50 passages, in comparison to the parental virus. MATERIAL AND METHODS: ASFV isolate Odintsovo 02/14 (parental), ASFV adapted variants ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 were all used to extract genomic DNA (gDNA). Sequencing library was constructed using the «Nextera XT DNA library preparation kit¼ («Illumina¼, USA). RESULTS: Genomes of ASFV/ARRIAH/CV-1/30 and ASFV/ARRIAH/CV-1/50 consisted of 186 529 bp and 186 525 bp, respectively. Total 78 single nucleotide polymorphisms (SNPs) were identified between the parental Odintsovo 02/14 and the two high passaged strains, as well as a 2947 bp large-size deletion in the 3' variable region of adapted viruses was detected. DISCUSSION: ASFV as a DNA-containing virus may not have a very high level of mutation, but this is the second study showing that adaptation to growth in continuous CC leads to large deletions in the genome of the virus. CONCLUSION: Mutations in the protein-coding regions of the genome can be synonymous and non-synonymous, i.e. leading to amino acid substitution. Additional research is needed to understand the influence of the mutations described in the adaptation process on the reproduction of the virus and its virulence.

[ASF virus replication features in the presence of recombinant proteins CD2v, pX69R and pE248R.]

INTRODUCTION: African swine fever (ASF), sever hemorrhagic disease of swine caused by a large DNA virus of the Asfaviridae family. Since there are no effective and safe vaccines against ASF yet, it is urgent to study the functions of its proteins, which is applicable by analyzing the features of ASF virus replication in the presence of recombinant proteins in vitro. PURPOSE: To study the effect of ASFV recombinant proteins CD2v, pE248R and pX69R on the speed and level of reproduction of ASF virus in vitro. Thus, obtain the necessary knowledge to develop approaches for creating a vaccine against ASF. MATERIALS AND METHODS: ASFV isolate Krasnodar 07/17 and strain ASF/ARRIAH/CV-1 were used. Cloning of X69R, EP402R, and E248R genes was performed in the pJET1.2 / blunt vector and pCI-neo in E. coli JM-109 cells, according to the manufacturer's manual. Localization of recombinant proteins in CV-1 cell line carried out by direct immunofluorescence reaction (DIF) using polyclonal antibodies conjugated to FITC. The ASF virus reproduction level was assessed by hemadsorption reaction and qPCR kit (Central Research Institute of Epidemiology). RESULTS: Recombinant plasmids pCI-neo / E248R, pCI-neo / EP402R and pCI-neo / X69R were constructed. The localization and the specificity of the obtained recombinant proteins CD2v, pE248R and pX69R was confirmed. It was established that these recombinant proteins induce the level of ASF virus reproduction on days 3-5 of the experiment by ~ 1.2-1.5 lgHADU50/cm3 in comparison with the negative control. DISCUSSION: The data obtained demonstrate the important role of CD2v, pX69R and pE248R proteins in the reproduction of the virus, since they significantly affect its level. The exact function of pX69R protein was not determined, however, in the experiments its positive effect on ASF virus reproduction was established, manifested in an increase in its reproduction level. CONCLUSION: This methodology allows us to study the nature of the effect of proteins with unknown function on ASF virus replication.

[Comparative research into sensitivity and specificity of immune-enzyme analysis with chemiluminescence and colorimetric detection for detecting antigens and antibodies to avian influenza viruses and newcastle disease].

The goal of this work was to demonstrate the results of the development of the enzyme-linked immunosorbent tests with chemiluminescence detection and colorimetric detection of specific viral antigens and antibodies for identifying the avian influenza and the Newcastle disease viruses: high sensitivity and specificity of the immuno- chemiluminescence assay, which are 10-50 times higher than those of the ELISA colorimetric method. The high effectiveness of the results and the automation of the process of laboratory testing (using a luminometer) allow these methods to be recommended for including in primary screening tests for these infectious diseases.

Spectroscopic study of biogenic amine complexes formed at fumed silica surface.

The structure and stability of biogenic amine complexes formed at the fumed silica surface were studied by UV-, IR-spectroscopy and TPD MS techniques. It was found that surface complexes are formed due to electrostatic interactions between amine cations and ionized silanol groups. The mechanism of thermal transformations of surface complexes is proposed. Kinetic parameters of thermal reactions at the fumed silica surface have been calculated.

[Biodegradable microcapsules containing DNA for the new DNA vaccine design].

A general method for the preparation of biodegradable microcapsules capable of antigen inclusion is suggested. Multilayer microcapsules were obtained by the method of level-by-level sorption of various polyelectrolytes (alginate, poly-L-lysine, kappa-carrageenan, and chitosan and dextran derivatives). High inclusion efficiency was found for protein and plasmid DNA (no less than 90%). A series of microcapsules with included pTKShi plasmid that incorporated a genome site encoding the E(2) polypeptide of the classic pig plague virus were obtained for carrying out in vivo experiments. It was shown that introduction to mice of microcapsules with the included pTKShi plasmid induced an immune response. The highest antibody titers of the mouse blood sera were obtained in immunization by microcapsules based on the modified dextran/carrageenan and modified chitosan/carrageenan. The method of antigen inclusion into biodegradable microcapsules could be used for the development of encapsulated vaccines of a new generation (DNA vaccines).

Morphology and surface properties of fumed silicas.

Several series of fumed silicas and mixed fumed oxides produced and treated under different conditions were studied in gaseous and liquid media using nitrogen and water adsorption-desorption, mass spectrometry, FTIR, NMR, thermally stimulated depolarization current (TSDC), photon correlation spectroscopy (PCS), zeta potential, potentiometric titration, and Auger electron spectroscopy methods. Aggregation of primary particles and adsorption capacity (Vp) decrease and hysteresis loops of nitrogen adsorption-desorption isotherms becomes shorter with decreasing specific surface area (S(BET)). However, the shape of nitrogen adsorption-desorption isotherms can be assigned to the same type independent of S(BET) value. The main maximum of pore size distribution (gaps between primary nonporous particles in aggregates and agglomerates) shifts toward larger pore size and its intensity decreases with decreasing S(BET) value. The water adsorption increases with increasing S(BET) value; however, the opposite effect is observed for the content of surface hydroxyls (in mmol/m2). Associative desorption of water (2(SiOH)-->SiOSi+H2O) depends on both the morphology and synthesis conditions of fumed silica. The silica dissolution rate increases with increasing S(BET) and pH values. However, surface charge density and the modulus of zeta-potential increase with decreasing S(BET) value. The PCS, 1H NMR, and TSDC spectra demonstrate rearrangement of the fumed silica dispersion depending on the S(BET) value and the silica concentration (C(SiO2)) in the aqueous suspensions. A specific state of the dispersion is observed at the C(SiO2) values corresponding to the bulk density of the initial silica powder.

[Potentialities of in vitro evaluation of the efficiency and safety of agents for devitalized tooth bleaching]. / Vozmozhnosti otsenki in vitro éffektivnosti i bezopasnosti sredstv dlia otbelivaniia devital'nykh zubov.

Pressing problems in bleaching devital teeth are discussed. Bleaching Endoperox and Brilliant were studied in vitro. For evaluating the safety of devital teeth bleaching, the microhardness of dental tissues was evaluated after intra-crown bleaching. The most significant changes in microhardness were observed in the coat dentin area.

The Structure and Properties of TiO(2)-Cu(II)-EDTA Ternary Surface Complexes.

The formation, composition, structure, and electrochemical properties of ternary surface complexes between copper(II) and ethylenediaminetetraacetate adsorbed on TiO(2) xerogels and on thin-film TiO(2) electrodes from solutions of varying pH have been studied by potentiometry, EPR spectroscopy, and electrochemical methods. The results strongly indicate that, in contrast to other organic ligands, B-type ternary surface complexes are formed in this system. The organic ligand forms an isolating layer between the surface of the TiO(2) electrode and the redox-active copper ions. Copyright 2001 Academic Press.

Formation of Ternary Surface Complexes by Adsorption of 2,2'-Bipyridine onto Silica Modified with Copper Ions.

The adsorption of 2,2'-bipyridine on a copper-containing silica surface has been studied. The equilibrium of bipyridine binding to surface copper ions are well reproduced by the constants for the formation of ternary surface complexes from an aqueous solution containing copper ions and bipyridine. This indicates clearly that under experimental conditions all reactions are reversible and speciation is controlled by thermodynamics. Copyright 2001 Academic Press.