[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.

[Antigenic and immunogenic activity of virus-like particles based on rabbit hemorrhagic disease virus (Caliciviridae: Lagovirus) genotypes GI1 and GI2 recombinant major capsid proteins].

INTRODUCTION: Rabbit hemorrhagic disease is an acute highly contagious infection associated with two genotypes of pathogenic Lagovirus. Antibodies to major capsid protein (Vp60) are protective. The aim of the work ‒ is an evaluation of antigenic and immunogenic activity of virus-like particles (VLPs) based on recombinant major capsid proteins of both genotypes of rabbit hemorrhagic disease virus (RHDV) (recVP60-GI1 and recVP60-GI2). MATERIALS AND METHODS: Baculovirus-expressed VLPs were evaluated using electron microscopy and administered to clinically healthy 1.53 month old rabbits in a dose of 50 g. Rabbits were challenged with 103 LD50 of virulent strains Voronezhsky-87 and Tula 21 days post immunization. Serum samples were tested for the presence of RHDV-specific antibodies. RESULTS: VLPs with hemagglutination activity forming VLP 3040 nm in size were obtained in Hi-5 cell culture. Specific antibody titers in rabbits measured by ELISA were 1 : 200 to 1 : 800 on 21th day post immunization with VLPs. Immunogenic activity of recVP60-GI1 VLPs was 90 and 40%, while it was 30 and 100% for recVP60-GI2 VLPs after the challenge with RHDV genotypes 1 and 2 respectively. The immunogenicity of two VLPs in mixture reached 100%. DISCUSSION: VLPs possess hemagglutinating, antigenic and immunogenic activity, suggesting their use as components in substances designed for RHDV specific prophylaxis in rabbits. Results of the control challenge experiment demonstrated the need to include the antigens from both RHDV genotypes in the vaccine. CONCLUSION: Recombinant proteins recVP60-GI1 and recVP60-GI2 form VLPs that possess hemagglutinating an antigenic activity, and provide 90100% level of protection for animals challenged with RHDV GI1 and GI2 virulent strains.

[Virus-like particles based on rotavarus A recombinant VP2/VP6 proteins for assessment the antibody immune response by ELISA].

INTRODUCTION: Rotavirus infection is one of the main concerns in infectious pathology in humans, mammals and birds. Newborn piglets or rodents are usually being used as a laboratory model for the evaluation of immunogenicity and efficacy for all types of vaccines against rotavirus A (RVA), and the use of ELISA for the detection of virus-specific antibodies of specific isotype is an essential step of this evaluation. OBJECTIVE: Development of indirect solid-phase ELISA with VP2/VP6 rotavirus VLP as an antigen to detect and assess the distribution of RVA-specific IgG, IgM and IgA in the immune response to rotavirus A. MATERIALS AND METHODS: VP2/VP6 rotavirus VLP production and purification, electron microscopy, PAGE, immunoblotting, ELISA, virus neutralization assay. RESULTS: The study presents the results of development of a recombinant baculovirus with RVA genes VP2-eGFP/VP6, assessment of its infectious activity and using it for VLP production. The morphology of the VP2/VP6 rotavirus VLPs was assessed, the structural composition was determined, and the high antigenic activity of the VLP was established. VLP-based ELISA assay was developed and here we report results for RVA-specific antibody detection in sera of different animals. CONCLUSION: The developed ELISA based on VP2/VP6 rotavirus VLP as a universal antigen makes it possible to detect separately IgG, IgM and IgA antibodies to rotavirus A, outlining its scientific and practical importance for the evaluation of immunogenicity and efficacy of traditional vaccines against rotavirus A and those under development.

[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.

[Problems of ante mortem diagnostics of prion diseases].

The review presents the state-of-the-art on the problem of diagnosis of prion diseases (PD) in humans and animals with a brief description of their etiology and pathogenesis. We pointed out that understanding the nature of the etio logical agent of PD determined their zoonotic potential and led to the development of highly specific immunological diagnostic methods aimed at identifying the infectious isoform of prion protein (PrPd) as the only marker of the disease. In this regard, we briefly summarize the results of studies, including our own, concerning the conversion of normal prion protein molecules (PrPc) to PrPd, the production of monoclonal antibodies and their application as immunodiagnostic reagents for the post-mortem detection of PrPd in various formats of immunoassay. We also emphasize the issues related to the development of methods for ante mortem diagnostics of PD. In this regard, a method for amplifying amino acid sequences using quacking-induced conversion of PrPc to PrPd in real time (RTQuIC) described in details. The results of recent studies on the assessment of the sensitivity, specificity and reproducibility of this method, carried out in various laboratories around the world, are presented. The data obtained indicate that RT-QuIC is currently the most promising laboratory assay for detecting PrPd in biological material at the preclinical stage of the disease. The significant contribution of US scientists to the introduction of this method into clinical practice on the model of diagnosis of chronic wasting disease of wild Cervidae (CWD) is noted. The possible further spread of CWD in the population of moose and deer in the territories bordering with Russia, as well as the established fact of alimentary transmission of CWD to macaques, indicate the threat of the appearance of PD in our country. In conclusion, the importance of developing new hypersensitive and/or selective components of known methods for PrPd identification from the point of view of assessing the risks of creating artificial infectious prion proteins in vivo or in vitro, primarily new pathogenic isoforms ("strains") and synthetic prions, was outlined.

[Assessment of immunogenic activity of the cloned human rotavirus A WA strain.]

INTRODUCTION: Rotovirus infection (RVI) caused by the dsRNA-containing virus from genus Rotavirus, Reoviridae family, belonging to group A (RVA), is the cause of severe diarrhea in human and other mammalian species. Vaccination is the most effective way to reduce the incidence of RVI. At present, the effectiveness of using gnotobiotic piglets as a universal model for reproducing human rotavirus infection and assessing the quality of RVI vaccine preparations has been experimentally proven. OBJECTIVES: Evaluation of immunogenic activity of the cloned RVA Wa strain in the new-born Vietnamese potbellied piglets trial. MATERIAL AND METHODS: Development of viral preparations of the cloned human Wa strain PBA, development of human RVA rVP6, ELISA, polymerase chain reaction with reverse transcription, immunization and experimental infection of newborn piglets. RESULTS: The article presents the results of the experiment on double immunization of newborn piglets with native virus preparations with the infection activity 5.5 lg TCID50/ml, 3 cm3 per dose, HRV with adjuvant 500 µg per dose and mock preparation (control group) followed with experimental inoculation of all animals with virulent virus strain Wa G1P[8] human RVA with infectious activity of 5.5 lg TCID50/ml in 5 cm3 dose. Development of clinical signs of disease and animal death were observed only in control group. RT-PCR system to detect RVA RNA in rectal swabs, samples of small intestine and peripheral lymph nodes was developed. ELISA based on obtained human RVA rVP6 was developed and results on RVA-specific IgG-antibodies in serum samples of experimental piglets are presented. CONCLUSION: In the course of the research, a high immunogenic activity of the native and purified virus of the cloned Wa RVA strain Wa was established and the possibility of its use as the main component of the RVI vaccine was confirmed. The possibility of using conventional newborn pigs instead of gnotobiotic piglets as an experimental model was demonstrated.

[Molecular and biological characteristics of vaccinary ERA-CB 20M of rabies virus.]

The molecular and biological characteristics of the vaccine against rabies virus strain ERA-CB 20M obtained by the Russian rabiologist, doctor of medical sciences S.V. Gribencha by adapting and cloning the strain ERA and SAD in a transplantable BHK-21 C13 cell culture are presented. The spectrum of the most sensitive strain of rabies ERA-CB 20M cell lines was determined and the level of glycoprotein was quantitatively determined. Primary nucleotide sequences of fragments of the genome of the strain ERA-CB 20M (genes N and G) were obtained and phylogenetic analysis was carried out. Molecular analysis showed that this strain belongs to the group of vaccine strains SAD1. When compared with the reference strain SAD1, 10% of the nucleotide differences were revealed in the gene fragment N; 15%, in the gene fragment G.

Diagnostic and vaccine chapter.

The first report in this chapter describes the development of a killed composite vaccine. This killed vaccine is non-infectious to humans, other animals, and the environment. The vaccine has low reactivity, is non-abortive, and does not induce pathomorphological alterations to the organs of vaccinated animals. The second report of this chapter describes the diagnostic value of a competitive enzyme-linked immunosorbent assay for detecting Brucella-specific antibodies and its ability to discriminate vaccinated cattle from infected cattle. The results indicated that the competitive enzyme-linked immunosorbent assay is more sensitive than traditional tests for detecting antibodies to Brucella abortus in naturally and experimentally infected cattle.

Epitope Specificity of Monoclonal Antibodies to Porcine Immunoglobulin G and Light Chains.

Monoclonal antibodies to porcine IgG and light chains were produced and characterized. The specificity of selected mAbs was revealed by their selective reactivity with porcine immunoglobulins in ELISA, immunodiffusion, immunoelectrophoresis, and immunoblot analysis. For determining the presence of different IgG and light chains epitopes responsible for cross-reactivity (i.e. common epitopes) on the heterologous immunoglobulins, all mAbs were tested using sera and IgG from 12 animal species and man. All mAbs were used in unlabeled and HRP-labeled form in competitive ELISA to map the antigenic structure of the IgG and L chains. Four different epitopes shared by isotypes IgG1 and IgG2, and therefore specific for IgG class determinants, were recognized. In addition, four different epitopes specific for IgG1 were identified. Three mAbs are directed to different L-chains of porcine immunoglobulins, 2 mAbs recognized slower-migrating light chain L1, whereas one mAb reacted with the faster-migrating light chain L2 in porcine immunoglobulins. For the detection and quantitation of IgG1, a sandwich ELISA was developed. The availability of the set of mAbs to porcine immunoglobulin isotypes will stimulate and facilitate a further study of the porcine immune system.

Determination of Cross-Reactive (Common) Epitopes of Animal and Human Immunoglobulins Using Monoclonal Antibodies.

Monoclonal antibodies to cross-reactive epitopes of IgG and IgM of some animal species and man were produced. Specificity and cross-reactivity of the monoclonal antibodies were determined by ELISA, immunoblotting, immunodiffusion and immunoelectrophoresis. The presence of immunoglobulin epitopes common to some animal species and man that were localized on both native Ig molecules (conformational) and polypeptide chains (linear) was demonstrated. The use of cross-reactive monoclonal antibodies for quantitation and qualitation of animal and human Ig in biological fluids by different immunoassays has been showed.

Thiophilic adsorption: rapid purification of F(ab)2 and Fc fragments of IgG1 antibodies from murine ascitic fluid.

A thiophilic adsorption method has been developed for rapid purification and separation of mouse F(ab)2 and Fc fragments obtained after proteolytic digestion of IgG1 monoclonal antibodies. Partially purified Mabs were digested with papain. Thiophilic chromatography was performed using stepwise elution with decreasing concentrations of ammonium sulphate. Most contaminating proteins did not react with the thiophilic adsorbent, and chromatography efficiently resolved the F(ab)2 and Fc fragments, as judged by electrophoresis. Fractions containing the F(ab)2 fragments retained about 90% of the total antibody activity loaded onto the column.