[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.

[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.

[Synthesis and characterization of human rotavirus A (Reoviridae: Sedoreovirinae: Rotavirus: Rotavirus A) virus-like particles].

INTRODUCTION: Rotavirus infection is the leading cause of acute gastroenteritis among infants. The development of new vaccines against rotavirus A is urgent because the virus has many genotypes, some of which have regional prevalence. Virus-like particles (VLP) is a promising way to create effective and safe vaccine preparations.The purpose of the study is to develop the technology for the production of VLP, containing VP2, VP4, VP6 and VP7 of viral genotypes prevalent on the territory of the Russian Federation, and to give its molecular genetic and virological characteristics. MATERIAL AND METHODS: The virulent strain Wa G1P[8] of human RV A adapted to MARC-145 cell culture has been used. It was cultured and purified according to the method described by the authors earlier. Standard molecular genetic and cytological methods were used: gene synthesis; cloning into transfer plasmids; recombinant baculoviruses production in Bac-to-Bac expression system; VLP production in the insect cells; centrifugation in sucrose solution; enzyme-linked immunosorbent assay (ELISA); electron microscopy (EM); polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. RESULTS: VP4 and VP7 of the six most represented in Russia genotypes: G1, G2, G4, G9, P4, P8, as well as VP2 and VP6 were selected for VLP production. Recombinant baculoviruses were obtained with codon frequencies optimized for insect cells. Cabbage loopper (Trichoplusia ni) cell culture was coinfected with different combinations of baculoviruses, and VLP consisting of 2-4 proteins were produced. VLP were purified by centrifugation. The size and morphology of the particles matched the rotavirus A virion (by EM). The presence of rotavirus A proteins in VLP was confirmed by the ELISA, SDS-PAGE and western blot analysis. CONCLUSION: The technology for the synthesis of three-layer VLP consisting of VP2, VP4, VP6 and VP7 has been developed and optimized. The resulting VLP composition represents 6 serotypes of VP4 and VP7, which are most represented on the territory of Russia, and can be used for vaccine development.

[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.

[Evaluation of the molecularbiological properties of human rotavirus A strain WA.]

BACKGROUND: Rоtaviruses are amоng the leading causes of severe diarrhea in children all over the Wоrld. Vaccination is considered to be the mоst effective way to cоntrоl the disease. Currently available vaccines for prevention of rоtavirus infection are based on live attenuated rotavirus strains human оr animal origin. OBJECTIVES: The aim of this investigation was to study the biological and genetic properties of an actual epidemic human rotavirus A (RVA) strain Wa G1P[8] genotype. METHODS: RVA Wa reproduction in a monolayer continuous cell lines, purification and concentration of RVA antigen, PAAG electrophoresis and Western-Blot, electrophoresis of viral genomic RNA segments, sequencing. RESULTS: Human RVA G1P[8] Wa strain biological and molecular genetic properties were assessed in the process of the adaptation to MARC145 continuous cell line. Cell cultured RVA antigen was purified, concentrated and then characterized by the method of PAAG electrophoresis and immunoblot. To verify RVA Wa genome identity, electrophoresis of viral genomic RNA segments was performed. The lack of accumulation of changes in the RVA Wa genome during adaptation to various cell cultures and during serial passages was demonstrated by sequencing fragments of the viral genome. CONCLUSIONS: RVA Wa strain is stable, it possesses high biological activity: it has been successfully adapted to the MARC145 cell line and RVA Wa virus titer after the adaptation reached 7,5-7,7 lg TCID50/ml. The identity of the cultivated RVA to the original strain Wa G1P[8] was confirmed.

[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.

Human rotavirus infection. Strategies for the vaccinal prevention.

Rotavirus was first isolated in 1973 in Australia from children with diarrhea. Hundreds of thousands of children die annually in developing countries from this virus with the mortality peaks in the most impoverished among them. According to wHo, rotavirus infection claims about 440 thousands children lives each year, being third in the mortality rate after pneumonia and malaria. Rotavirus is widely spread throughout the world and by the age of five years almost every child encountered this pathogen at least once. Rotavirus has a high genetic and antigenic diversity. The most important for humans is the group A rotavirus, and the most common by far genotypes are G1P [8], G2P [4], G3P [8], G4P [8], G9P [8], and to a lesser extent G12P [8]. There are three gene constellations described in rotavirus designated Wa, Ds-1, and Au-1. It is believed that they originated from rotaviruses of pigs, cattle, dogs, and cats, respectively. Cases of rotavirus interspecies transmission from animal to humans were reported. The first vaccines against rotavirus infection were based on naturally attenuated virus of the animal origin. Their efficiency, especially in developing countries, was inadequate, but today China and India use vaccines based on animal rotaviruses. Using the method of gene reassortation with the cattle rotavirus WC3 as a backbone, pentavalent vaccine against most common human rotavirus serotypes was developed and now successfully used as RotaTeq. The ability of rotavirus to protect against heterologous isolates was taken into account in the development of other vaccine, Rotarix, created on the basis of rotavirus genotype G1P1A [8]. The efficacy of these vaccines in developing countries is significantly reduced (51%), the cost of a dose is high, and so the search for more effective, safe, and inexpensive vaccines against rotavirus continues around the world.

[Enzyme immunoassay for detection of porcine circovirus type 2, by using the recombinant capsid protein ORF-2].

Recombinant antigen ORF2 from porcine circovirus type 2 (PCV-2) was produced, by using the baculovirus expression system, with histidine tags to allow purification by metal-chelate affinity chromatography. The purity of the protein was verified by polyacrylamide gel electrophoresis; and its immunospecificity was confirmed by the immunoblotting test using reference PCV-2-positive and PCV-2-negative porcine sera and monoclonal antibodies. The protein was used as an antigen to develop an indirect enzyme immunoassay (EIA) of PCV-2 antibodies. EIA was shown to have a high sensitivity and specificity as compared with indirect immunofluorescence test. Porcine serum samples from 15 pig-breeding farms of the Russian Federation were studied. Seropositive samples were found in all age pig groups in all the farms, The number of seropositive animals was shown to be directly related to its age.

Isolation and characterization of full-length recombinant cattle PrPC protein.

Full-length Bos taurus PrPC protein was obtained in the eu- and prokaryotic expression systems. Immunoblotting and indirect enzyme immunoassay demonstrated high specificity and antigenic activity of full-length proteins in the reactions with monoclonal antibodies (anti-SAF-32 and VRQ-84). Membrane location of recombinant PrPC protein in insect cells was shown by immunofluorescent analysis.

[Synthesis of recombinant proteins from the American and European viruses of the porcine reproductive and respiratory syndrome in baculovirus expression system and their antigenic properties]. / Sintez v bakulovirusnoi sisteme rekombinantnykh belkov amerikanskogo i evropeiskogo tipov virusa reproduktivnogo i respiratornogo sindroma svinei i ikh antigennye svoistva.

The baculovirus expression system was made use of to derive the recombinant nucleocapsid (N) proteins of the American and European virus types and of the porcine reproductive and respiratory syndrome (PRRS). The obtained products were purified by metal-affine chromatography and their specificity was confirmed in immunechemical reactions with reference monoclonal antibodies. The antigenic activity of recombinant proteins was studied by indirect immune enzyme assay (IEA) with porcine serum, which had been in advance characterized by the "HerdCheck" kit (IDEXX Co.). It was shown as possible to apply the derived recombinant antigens in determining, by indirect IEA, antibodies to the PRRS virus.