Electrochemical profiling of poliovirus particles inactivated by chemical method and ionizing radiation.

Electrochemical profiling of formaldehyde-inactivated poliovirus particles demonstrated a relationship between the D-antigen concentration and the intensity of the maximum amplitude currents of the poliovirus samples. The resultant signal was therefore identified as electrochemical oxidation of the surface proteins of the poliovirus. Using registration of electrooxidation of amino acid residues of the capsid proteins, a comparative electrochemical analysis of poliovirus particles inactivated by electrons accelerated with doses of 5 kGy, 10 kGy, 15 kGy, 25 kGy, 30 kGy at room temperature was carried out. An increase in the radiation dose was accompanied by an increase in electrooxidation signals. A significant increase in the signals of electrooxidation of poliovirus capsid proteins was detected upon irradiation at doses of 15-30 kGy. The data obtained suggest that the change in the profile and increase in the electrooxidation signals of poliovirus capsid proteins are associated with an increase in the degree of structural reorganization of surface proteins and insufficient preservation of the D-antigen under these conditions of poliovirus inactivation.

[The role of the encephalomyocarditis virus type 1 proteins L and 2A in the inhibition of the synthesis of cellular proteins and the accumulation of viral proteins during infection].

INTRODUCTION: Infection of cells with encephalomyocarditis virus type 1 (EMCV-1, Cardiovirus A: Picornaviridae) is accompanied by suppression of cellular protein synthesis. The main role in the inhibition of cellular translation is assigned to the L and 2A «security¼ proteins. The mechanism of the possible influence of the L protein on cellular translation is unknown. There are hypotheses about the mechanism of influence of 2A protein on the efficiency of cap-dependent translation, which are based on interaction with translation factors and ribosome subunits. However, the available experimental data are contradictory, obtained using different approaches, and do not form a unified model of the interaction between the L and 2A proteins and the cellular translation machinery. AIM: To study the role of L and 2A «security¼ proteins in the suppression of translation of cellular proteins and the efficiency of translation and processing of viral proteins in infected cells. MATERIALS AND METHODS: Mutant variants of EMCV-1 were obtained to study the properties of L and 2A viral proteins: Zfmut, which has a defective L; Δ2A encoding a partially deleted 2A; Zfmut&Δ2A containing mutations in both proteins. Translational processes in infected cells were studied by Western-blot and the pulse method of incorporating radioactively labeled amino acids (14C) into newly synthesized proteins, followed by radioautography. RESULTS: The functional inactivation of the 2A protein does not affect the inhibition of cellular protein synthesis. A direct correlation was found between the presence of active L protein and specific inactivation of cellular protein synthesis at an early stage of viral infection. Nonspecific suppression of the translational processes of the infected cell, accompanied by phosphorylation of eIF2α, occurs at the late stage of infection. Partial removal of the 2A protein from the EMCV-1 genome does not affect the development of this process, while inactivation of the L protein accelerates the onset of complete inhibition of protein synthesis. Partial deletion of the 2A disrupts the processing of viral capsid proteins. Suppression of L protein functions leads to a decrease in the efficiency of viral translation. CONCLUSION: A study of the role of EMCV-1 L and 2A proteins during the translational processes of an infected cell, first performed using infectious viral pathogens lacking active L and 2A proteins in one experiment, showed that 2A protein is not implicated in the inhibition of cellular translation in HeLa cells; L protein seems to play an important role not only in the specific inhibition of cellular translation but also in maintaining the efficient synthesis of viral proteins; 2A protein is involved not only in primary but also in secondary processing of EMCV-1 capsid proteins.

[Investigation of oncolytic potential of vaccine strains of yellow fever and tick-borne encephalitis viruses against glioblastoma and pancreatic carcinoma cell lines].

INTRODUCTION: Flaviviruses, possessing natural neurotropicity could be used in glioblastoma therapy using attenuated strains or as a delivery system for antitumor agents in an inactivated form. OBJECTIVE: To investigate the sensitivity of glioblastoma and pancreatic carcinoma cell lines to vaccine strains of yellow fever and tick-borne encephalitis viruses. MATERIALS AND METHODS: Cell lines: glioblastoma GL-6, T98G, LN-229, pancreatic carcinoma MIA RaCa-2 and human pancreatic ductal carcinoma PANC-1. Viral strains: 17D yellow fever virus (YF), Sofjin tick-borne encephalitis virus (TBEV). Virus concentration were determined by plaque assay and quantitative PCR. Determination of cell sensitivity to viruses by MTT assay. RESULTS: 17D YF was effective only against pancreatic carcinoma tumor cells MIA Paca-2 and had a limited effect against PANC-1. In glioblastoma cell lines (LN229, GL6, T98G), virus had no oncolytic effect and the viral RNA concentration fell in the culture medium. Sofjin TBEV showed CPE50 against MIA Paca-2 and a very limited cytotoxic effect against PANC-1. However, it had no oncolytic effect against glioblastoma cell lines (LN229, T98G and GL6), although virus reproduction continued in these cultures. For the GL6 glioblastoma cell line, the viral RNA concentration at the level with the infection dose was determined within 13 days, despite medium replacement, while in the case of the LN229 cell line, the virus concentration increased from 1 × 109 to 1 × 1010 copies/ml. CONCLUSION: Tumor behavior in organism is more complex and is determined by different microenvironmental factors and immune status. In the future, it is advisable to continue studying the antitumor oncolytic and immunomodulatory effects of viral strains 17D YF and Sofjin TBEV using in vivo models.

Perspectives for the creation of a new type of vaccine preparations based on pseudovirus particles using polio vaccine as an example.

Traditional antiviral vaccines are currently created by inactivating the virus chemically, most often using formaldehyde or ß-propiolactone. These approaches are not optimal since they negatively affect the safety of the antigenic determinants of the inactivated particles and require additional purification stages. The most promising platforms for creating vaccines are based on pseudoviruses, i.e., viruses that have completely preserved the outer shell (capsid), while losing the ability to reproduce owing to the destruction of the genome. The irradiation of viruses with electron beam is the optimal way to create pseudoviral particles. In this review, with the example of the poliovirus, the main algorithms that can be applied to characterize pseudoviral particles functionally and structurally in the process of creating a vaccine preparation are presented. These algorithms are, namely, the analysis of the degree of genome destruction and coimmunogenicity. The structure of the poliovirus and methods of its inactivation are considered. Methods for assessing residual infectivity and immunogenicity are proposed for the functional characterization of pseudoviruses. Genome integrity analysis approaches, atomic force and electron microscopy, surface plasmon resonance, and bioelectrochemical methods are crucial to structural characterization of the pseudovirus particles.

Properties and Activity of Peptide Derivatives of ACE2 Cellular Receptor and Their Interaction with SARS-CoV-2 S Protein Receptor-Binding Domain.

The aim of this work was to design and characterize peptides based on the α-helices h1 and h2 of the ACE2 receptor, forming the interaction interface between the receptor-binding domain (RBD) of the SARS-CoV-2 S protein and the cellular ACE2 receptor. Monomeric and heterodimeric peptides connected by disulfide bonds at different positions were synthesized. Solubility, RBD-binding affinity, and peptide helicity were experimentally measured, and molecular dynamics simulation was performed in various solvents. It was established that the preservation of the helical conformation is a necessary condition for the binding of peptides to RBD. The peptides have a low degree of helicity and low affinity for RBD in water. Dimeric peptides have a higher degree of helicity than monomeric ones, probably due to the mutual influence of helices. The degree of helicity of the peptides in trifluoroethanol is the highest; however, for in vitro studies, the most suitable solvent is a water-ethanol mixture.

Basic and Applied Sciences: Technology and Immunobiological Products.

The experience in organizing the creation of innovative immunobiological drugs and vaccines is discussed-from laboratory developments to industrial technologies and the registration of finished forms of effective and safe drugs that are in demand in the domestic and foreign markets. The principles of their action rely on the latest global achievements in the field of immunobiology and vaccinology.

Peptide Inhibitors of the Interaction of the SARS-CoV-2 Receptor-Binding Domain with the ACE2 Cell Receptor.

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin-converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22-23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which terminal cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of each precursor peptide molecule. The precursors h1 and h2 mimic amino acid sequences of α1- and α2-helices of the ACE2 extracellular peptidase domain, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their constituent peptides with RBD (particularly in dependence of the middle and terminal methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified to 95-97% purity, and characterized by HPLC and MALDI-TOF mass spectrometry. Binding of these peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. The original RBD of the Chinese variant bound to three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant. The antiviral activity of the proposed peptides was evaluated in Vero cell line.

[Peptide inhibitors of the interaction of the SARS-CoV-2 receptor-binding domain with the ACE2 cell receptor]. / Peptidnye ingibitory vzaimodeistviia retseptor-sviazyvaiushchego domena virusa SARS-CoV-2 s kletochnym retseptorom ASE2.

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22-23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which edge cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of the molecule of each of the precursor peptides. The precursors h1 and h2 modelled amino acid sequences of α1- and α2-helices of the extracellular peptidase domain of ACE2, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their RBD-peptides (particularly in dependence of the middle and edge methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified (to 95-97% purity), and characterized by HPLC and MALDI-TOF mass spectrometry. The binding of the peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. Binding to the original RBD of the Chinese variant was detected in three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant with micromolar constants. The antiviral activity of the proposed peptides in Vero cell culture was also evaluated.

[Real-time PCR assay development for the control of vaccine against hemorrhagic fever with renal syndrome].

INTRODUCTION: Hemorrhagic fever with renal syndrome (HFRS) holds a leading place among natural focal human diseases in Russian Federation. There is no etiotropic therapy for the disease now. The vaccine prophylaxis is the most effective method to control this infection. The main criteria for inactivated vaccines evaluation are its immunogenicity and specific activity.The study purposes were to develop a sensitive and specific real-time PCR method for viral RNA quantification in the inactivated vaccine and to study the correlation between the viral RNA amount and vaccine immunogenicity. MATERIAL AND METHODS: L-segment fragments of the Puumala, Hantaan, and Sochi vaccine strains were selected as diagnostic targets for oligonucleotides and fluorescent probes. The immunogenicity of experimental vaccines was determined by the induction of neutralizing antibodies in BALB/c mice. RESULTS: A highly specific, sensitive and reproducible real-time PCR method has been developed. The analytical sensitivity was 1.24 ± 1.5 x 102 copies/ml for Puumala virus; 1.16 ± 1.4 * 102 copies/ml for Hantaan; 1.32 ± 1.8 * 102 copies/ ml for Sochi, with a virus content of 1.5 ± 0.5 lg FFU/ml; 1.8 ± 0.5 lg FFU/ml and 2.2 ± 0.5 lg FFU/ml, respectively. The viral RNA amount in experimental vaccine preparations inactivated with ß-propiolactone was proportional to the neutralizing antibodies titer observed in mice following the immunization. DISCUSSION: It was found that different virus inactivators differently affects the detected viral RNA amount, but not the vaccine immunogenicity, which indicates the same degree of the immunogenic proteins damage. The direct relationship between the viral RNA copy number and vaccine immunogenicity makes it possible to use this criterion for vaccine dosage preparation. CONCLUSION: The developed method for viral RNA quantification is a promising tool for the specific activity control of the HFRS vaccine.

Evaluation of the Antiviral Potential of Modified Heterocyclic Base and 5'-Norcarbocyclic Nucleoside Analogs Against SARS-CoV-2.

The pandemic caused by the novel betacoronavirus SARS-CoV-2 has already claimed more than 3.5 million lives. Despite the development and use of anti-COVID-19 vaccines, the disease remains a major public health challenge throughout the world. Large-scale screening of the drugs already approved for the treatment of other viral, bacterial, and parasitic infections, as well as autoimmune, oncological, and other diseases is currently underway as part of their repurposing for development of effective therapeutic agents against SARS-CoV-2. In this work, we present the results of a phenotypic screening of libraries of modified heterocyclic bases and 5'-norcarbocyclic nucleoside analogs previously synthesized by us. We identified two leading compounds with apparent potential to inhibit SARS-CoV-2 replication and EC50 values in a range of 20-70 µM. The structures of these compounds can be further optimized to develop an antiviral drug.

[History of the discovery and study of tick-borne encephalitis in Russia: three Far Eastern expeditions (1937-1939)].

The review provides a brief historical outline of the discovery and study of tick-borne encephalitis (TBE) during three expeditions to the Far East (19371939). As a result of the Far Eastern expeditions, the TBE virus was discovered, numerous strains were isolated, a vector-borne transmission pathway was established, the main features of epidemiology, clinic and pathomorphology of the disease were described, serotherapy was tested, first inactivated "brain" vaccine against TBE was developed and its effectiveness was proved. The history of the discovery and study of TBE is marked by the heroism of researchers and tragic events illness of some participants, death during the development of the first vaccine, arrest of L.A. Zilber and two performers.

The Effect of a Lipopolysaccharide from Rhodobacter capsulatus PG on Inflammation Caused by Various Influenza Strains.

The development of a specific inflammation in mice that had been infected by two influenza virus strains, A/chicken/Kurgan/5/2005 (H5N1) and A/Hamburg/2009 MA (H1N1), was studied. We investigated the effect of a non-toxic lipopolysaccharide from Rhodobacter capsulatus PG on the survival and body weight of the mice, production of IgG antibodies, and the induction of pro- and anti-inflammatory cytokines in blood serum. The administration of the R. capsulatus PG lipopolysaccharide was shown to induce interferon-ß synthesis, both in healthy and influenza A virus-infected mice, and to promote production of antiviral antibodies in the blood of the influenza-infected animals.

[Hemorrhagic fever with renal syndrome group outbreak caused by Sochi virus.]

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) occupies a leading place among natural focal human diseases in the Russian Federation. Sporadic incidence of HFRS-Sochi has been annually recorded in the Krasnodar Territory since 2000. The group outbreak of the HFRS-Sochi was first registered in Gelendzhik in the fall of 2013. METHODS: Serological methods were used: indirect immunofluorescence, enzyme immunoassay, FRNT in Vero cells, and methods for the viral RNA detection: PCR and RT-PCR. RESULTS: Data of clinical, epidemiological, immunological and molecular studies of 3 out of 4 cases in HFRS-Sochi outbreak are presented. Severity of the disease correlated with early gastrointestinal disorders appearance. Patient MA gastrointestinal disorders were joined on day 3 of a fever. Clinical and laboratory studies revealed signs of kidneys, liver, pancreas damage, bilateral hydrothorax, bilateral polysegmental pneumonia and polyneuropathy. As a result of long-term treatment, the patient recovered. Patient AA had gastrointestinal disturbances the next day after fever onset. The patient was not saved, despite early hospitalization. Hantavirus antigen and RNA were detected in the lung tissues 2 out of 10 Black-Sea field mice captured in the affected area, as well as in the organs of deceased patient. The most severe clinical course of the disease in close relatives, son and father, with a fatal outcome in the latter case may be the result of genetic features. The severity and outcome of the disease was not depend on day of hospitalization and correlated with the early manifestations of gastrointestinal disorders. CONCLUSIONS: Presented data confirm high virulence and pantropism of the Sochi virus, as well as the epidemiological role of Black-Sea field mouse (Apodemus ponticus) as the host of the Sochi virus and the source of human infection.

Immunogenicity and safety of the adult tbe vaccine «tick-e-vac¼.

About 3,000 cases of TBE are registered annually in the Russian Federation. Vaccination is the main way to prevent the tick-borne encephalitis disease. Comparative study of the reactogenicity and immunogenicity of a new vaccine «Tick-E-Vac¼ was held. Volunteers aged from 16 years old were twice immunized with the vaccines «Tick-E-Vac¼ or «Encevir¼ derived from strains of Far East subtype of TBE virus, according to standard and emergency schemes. The clinical study was randomized, comparative, blind, and controlled. The frequency, intensity, time of occurrence, and duration of local and general reactions had been recorded. The titers of antiviral antibodies in ELISA had been determined to assess the immunological efficacy of vaccination. According to the results of the clinical study, the severity of local and general reactions in initial seronegative recipients was weak or moderate. The symptoms were usually manifested within 1-2 days after injection and persisted for not more than 4 days, after which time the symptoms disappeared. There was no statistically significant difference in the reactogenicity of the vaccines after the first and after the second injection. The reactogenicity also did not depend on the gender of recipients. After the first immunization, the level of seroprotection was not less than 43%; the average geometric titer of antibodies (GTA), not less than 1:200. After the second injection, the level of seroprotection reached 90-100%; GTA, not less than 1:500. The data on the reactogenicity and immunogenicity to the original seropositive recipients is not significantly different from the data for the initial seronegative recipients. The data indicate weak reactogenicity of the vaccines «Tick-E-Vac¼ and «Encevir¼. Double vaccination with an interval of 14 or 30 days leads to the formation of expressed immune response. Thus, differences in the level of seroprotection and in antiviral titers in the cases of the standard and emergency vaccination schedules are not statistically significant. The correlation between the development in recipients of local and general symptoms and the immunological efficacy of the vaccines has not been identified.

Genetic description of a tick-borne encephalitis virus strain Sofjin with the longest history as a vaccine strain.

Vaccines based on the strain Sofjin of the Far-Eastern tick-borne encephalitis virus (TBEV) subtype have been used for TBE prophylaxis for over 50 years in Russia and neighboring countries. On the wide territory, where all known TBEV subtypes are circulating, the cultural, purified, concentrated, inactivated TBE vaccine Moscow has been shown to be safe and efficacious in a massive immunization. In the present work, we describe the genome of the vaccine strain Sofjin. We have shown that it differs from TBEV strains previously published with the name "Sofjin". Moreover, we have shown the stability of the virus during the vaccine manufacturing process on the molecular level.