Development, production and characterization of SARS-CoV-2 virus-like particles (Coronaviridae: Orthocoronavirinae: Betacoronavirus: Sarbecovirus).

INTRODUCTION: The COVID-19 pandemic caused by SARS-CoV-2 has created serious health problems worldwide. The most effective way to prevent the occurrence of new epidemic outbreaks is vaccination. One of the modern and effective approaches to vaccine development is the use of virus-like particles (VLPs). The aim of the study is to develop a technology for production of VLP based on recombinant SARS-CoV-2 proteins (E, M, N and S) in insect cells. MATERIALS AND METHODS: Synthetic genes encoding coronavirus proteins E, M, N and S were used. VLP with various surface proteins of strains similar to the Wuhan virus, Delta, Alpha and Omicron were developed and cloned into the pFastBac plasmid. The proteins were synthesized in the baculovirus expression system and assembled into VLP in the portable Trichoplusia ni cell. The presence of insertion in the baculovirus genome was determined by PCR. ELISA and immunoblotting were used to study the antigenic activity of VLP. VLP purification was performed by ultracentrifugation using 20% sucrose. Morphology was assessed using electron microscopy and dynamic light scattering. RESULTS: VLPs consisting of recombinant SARS-CoV-2 proteins (S, M, E and N) were obtained and characterized. The specific binding of antigenic determinants in synthesized VLPs with antibodies to SARS-CoV-2 proteins has been demonstrated. The immunogenic properties of VLPs have been studied. CONCLUSION: The production and purification of recombinant VLPs consisting of full-length SARS-CoV-2 proteins with a universal set of surface antigens have been developed and optimized. Self-assembling particles that mimic the coronavirus virion induce a specific immune response against SARS-CoV-2.

[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.

[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.

Enhanced expression of trim14 gene suppressed Sindbis virus reproduction and modulated the transcription of a large number of genes of innate immunity.

In the present research, we have studied an influence of enhanced expression TRIM14 on alphavirus Sindbis (SINV, Togaviridae family) infection. In the HEK293 cells transfected with human trim14 gene (HEK-trim14), SINV yield after infection was decreased 1000-10,000 times (3-4 lg of TCD50/ml) at 24 h p.i. and considerably less (1-2 lg of TCD50/ml) at 48 h p.i. Analysis of the expression of 43 genes directly or indirectly involved in innate immune machine in HEK-trim14 non-infected cells comparing with the control (non-transfected) HEK293 cells revealed that stable trim14 transfection in HEK293 cells caused increased transcription of 18 genes (ifna, il6 (ifnß2), isg15, raf-1, NF-kB (nf-kb1, rela, nf-kb2, relb), grb2, grb3-3, traf3ip2, junB, c-myb, pu.1, akt1, tyk2, erk2, mek2) and lowered transcription of 3 genes (ifnγ, gata1, il-17a). The similar patterns of genes expression observe in SINV-infected non-transfected HEK293 cells. However, SINV infection of HEK-trim14 cells caused inhibition of the most interferon cascade genes as well as subunits of transcription factor NF-κB. Thus, stable enhanced expression of trim14 gene in cells activates the transcription of many immunity genes and suppresses the SINV reproduction, but SINV infection of HEK-trim14 cells promotes inhibition of some genes involved in innate immune system.

[Engineering by reverse genetics and characterization of the new reassortant influenza virus strain H5N1].

Reverse genetics was applied to engineering of the reassortantvaccine candidate strain against highly pathogenic avian influenza viruses (HPAIVs) of the H5 subtype. The new strain recPR8-H5N1 contains the HA gene from the Russian HPAIV A/Kurgan/05/2005 (H5N1), the NA and internal genes from A/Puerto Rico/8/34 (H1N1). The strain recPR8-H5N1 demonstrated the antigenic specificity (H5), high proliferation rate in 12 days chicken embryos, and was lethal for the embryos in 36 hours. An inactivated emulsified vaccine based on the strain recPR8-H5N1 elicited high antibody titers and protected 6-week-old chickens from lethal challenge with the HPAIV A/Kurgan/05/2005 (H5N1) on day 21 after single immunization. Infection of non-vaccinated birds with the strain recPR8-H5N1 did not cause any pathology, and the virus was not detected using PCR in blood and cloacal swabs on day 7 p.i. Specific weak seroconversion caused by infection with the strain recPR8-H5N1 was detected on day 14 p.i. As a result, a new influenza virus strain was obtained with modified properties.

[HIV-1 assembly is initiated by p17 matrix protein].

HIV-1 matrix protein (MA) is multifunctional structural protein located on N-terminus of Gag precursor p55 and responsible for its transport to plasma membrane, the site of virus assembly. Here, it has been shown that MA is cleaved from Gag precursor at early stage of the virus infection and participates in virus assembly. MA is transported into the nuclei wherein it associates with viral RNA (vRNA). The MA-vRNA complex is transported to plasma membrane. Mutant MA which lost its membranotropic signal does not reach plasma membrane and MA-vRNA complex remains in the nuclei and cytoskeleton. Thus, MA seems to deliver vRNA from the nuclei to plasma membrane through cytoskeleton initiating virus assembly.

[The maturation steps of human immunodeficiency virus and the role of proteolysis].

HIV-1 virions are as immature noninfectious particles lacking a central core. Shortly after budding, virions temporally mature and acquire cores and infectious activity. The cause of maturation remains poorly studied. We have revealed that the virions produced early after infection following 24-36 hours, never mature and remain noninfectious, and only virions produced 48-72 hours after infection mature. The mature virions contain 3 times more genomic viral RNA than "early" virus. The "early" virions contain the same proteolytically cleaved Gag proteins as mature virions in contrast to the accepted version. The virus protease inhibitor Indinavir sulfate (IS) fully blocks infectivity when added early after infection. The early proteolysis of Gag precursor in the infected cells and inclusion into the virions of cellularly cleaved matrix protein (cMA) are shown in the IS-treated cells. cMA is associated with genomic viral RNA.

[Effects of novel antiviral agents on HIV-1 replication].

Two groups of the antiviral agents: 1) adamantane- and norbornen-containing compounds with in-built cholesterol to potentiate the membranotropic properties and 2) synthetic matrix protein peptides (peptides A and B) were found to have effects on HIV replication. The agents of the former group produced antiviral activity only when added in combination with the virus. Peptide A (matrix protein 43-60 amino acids) inhibited viral replication when added in both the early and late periods. Fluorescein-labeled peptide A was detectable in the cytoplasm and nucleus (although adsorption of a portion of the peptides cannot be excluded onto the cell surface). Peptide A was shown to inhibit Gag precursor p55 transport from the nuclei to the plasma membrane, the site of virus assembly. Peptide B had no antiviral activity.

[Assembly and maturation of HIV-1--targets for new anti-HIV compounds].

Much progress has been recently made in research of the final stages of the HIV-1 life cycle, i.e. of its assembly, gemmation and maturation. The virus was shown, in particular, to use widely cell mechanisms in its replication and assembly. The TSG 101 cellular endosomal sorting protein interacting with the p6 viral protein is necessary for gemmation. Cyclophilin and HP68 (cell proteins) needed for marphogenesis of the virus were identified. The recently obtained data on the interaction of Vif (a viral protein) and Apobec (a cell protein) showed that HIV-1 has an action mechanism overcoming the cell barriers. The "early" virus phenomenon, which is deprived of any mature structure or the ability to infect and does not contain mature Gag and Env proteins, illustrates that the proteolytic pressing of the Gag p55 precursor is not enough for the maturation of the virus and additional viral or cellular factors are needed for the virus to become infectious. Although the current antiviral therapy has been successful enough, it is far from being effective in all cases; one of the reasons is resistance to chemodrugs developing rapidly in patients. Fast mutations and exceptional plasticity of the viral genome (which helps the virus to develop rapidly resistance to drugs) belong to the major problems. The circulation of persistent virus variants has been quickly increasing. There is an urgent need in developing new antiviral drugs acting on new viral targets; progress in experimental virology would speed it up. Thus, new drugs can be created, which block the activity of Vif, that would make Apobec block the virus replication. Compounds can be developed, which block the interaction of cyclophilin and TSG101 with viral proteins. The recently described importance of cholesterol in the sexual transfer of viruses is expected to bring simple and inexpensive compounds destroying cholesterol in the mucous tunic of genitals into clinical use. The identification of additional factors needed for the maturation of the virus and for its becoming infectious can be a basis for the development of drugs blocking their packaging into virions. Future research is expected to define new targets for the chemotherapy of AIDS and to promote the designing of new chemodrugs.

[HIV-1 p17 matrix protein is transported into the cell nucleus and binds with genomic viral RNA]. / Matriksnyi belok p17 VICh-1 transportiruetsia v kletochnoe iadro i sviazyvaetsia s genomnoi virusnoi RNK.

We have shown that gag polyprotein p55 is cleaved in cytosol rapidly after its synthesis, during 2 h, and p17 enters the nuclei while p24 resides in cytosol. To determine whether the nascent p17 is associated with viral genomic RNA in the nuclei, the cells were fractionated, the viral complexes were immunoprecipitated by monoclonal antibodies against gag proteins, and RNA was extracted and analyzed by slot and blot hybridization. Monoclonal antibodies against p17 precipitated all the viral RNA from the nuclei including full-size genomic RNA and essential part from membranes while monoclonal antibodies against p24 did not precipitate any viral RNA from the nuclei. These data suggest that matrix protein is linked to genomic RNA in the nuclei and rise the possibility that p17 may transfer viral nucleocapsids from the nuclei to plasma membranes, the site of virus assembly.

HIV-1 matrix protein p17 resides in cell nuclei in association with genomic RNA.

We have shown previously that HIV-1 matrix protein p17 is transported to the nucleus of Jurkat-tat and H9 cells soon after infection. As shown in this combination, gag polyprotein p55 synthesized 48 h after cell infection is cleaved in cytosol rapidly after its synthesis, and nascent p17 enters the nuclei and gradually accumulates there. Uncleaved p55 molecules and intermediate precursors are rapidly transported to the membranes and are also found in nuclei. Mature gag proteins are seen in membranes only after prolonged period of labelling or chase (4 or more hours later). To determine whether the nascent p17 is associated with viral genomic RNA in the nuclei, the cells were fractionated, the viral complexes were immunoprecipitated by monoclonal antibodies (MAbs) against gag proteins, and RNA was extracted and analyzed by slot and blot hybridization. MAb against p17 precipitated all the viral RNA from the nuclei including full-size genomic RNA and essential parts from membranes while MAb against p24 did not precipitate any viral RNA from the nuclei. These data suggest that matrix protein is linked to genomic RNA in the nuclei and raise the possibility that p17 may transfer viral nucleocapsids from the nuclei to plasma membranes, the site of virus assembly.

[Conservativism of the matrix protein of paramyxoviruses]. / Konservatizm matriksnogo belka paramiksovirusov.

Cross reactions among paramyxoviruses were determined by the immunoblot method. Human parainfluenza viruses, types 1-3, avian parainfluenza virus type 4, mumps, Sendai, and measles viruses were used. Antisera to human parainfluenza viruses were shown to cross-interact with proteins NP and M of other types, and all antisera to the members of Paramyxovirus genus cross-reacted with M proteins of other paramyxoviruses. No cross reactions with measles virus proteins were observed. It is concluded that M protein is the most conservative protein of paramyxoviruses.

[Use of an immune blotting method (western) for studying viral antigens and for detecting antibodies to viral proteins]. / Primenenie metoda immunnogo ("vestern") blotinga dlia izucheniia virusnykh antigenov i vyiavleniia antitel k virusnym belkam.

The technique of the procedure and the results of the use of immune ("western") blotting method for studies on viral antigens and detection of antibodies to individual proteins of viral particles are described. The possibility of detection and study of individual viral antigens in the whole plasma of patients or carriers is demonstrated by the example of HBs antigen of human hepatitis B virus. The method of immune blotting was used for screening of human sera for the detection of antibodies to the AIDS virus proteins. The sera under study positive for antibodies to AIDS virus by preliminary solid-phase enzyme-immunoassay were shown to contain actually the antibodies to different proteins of AIDS virus. The antibody levels to individual AIDS virus proteins varied in different sera. Some sera positive for antibodies to AIDS virus by the solid-phase enzyme-immunoassay contained no antibody to AIDS virus proteins but reacted with cellular proteins present in the antigen. The immune blotting method was also used for determinations of the spectrum of antibodies in animal sera produced by immunization with different viral antigens.

[Rapid diagnosis of influenza: a comparative evaluation of different methods]. / Bystraia diagnostika grippa: sravnitel'naia otsenka razlichnykh metodov.

The results of influenza diagnosis during the outbreak of 1985 are presented. Nasopharyngeal secretions from 94 patients were examined by virus isolation in chick embryos, fluorescent antibody technique (FAT). enzyme-immunoassay (EIA), and dot-blot hybridization method (DBHM). The virus was isolated in 28%, FAT was positive in 22%, EIA in 47% of the cases. Among 94 secretion specimens 40 were tested by DBHM. In this instance, virus was isolated in 37%, EIA was positive in 65%, and DBHM in 85% of the cases. It seems advisable to use EIA based on the detection of the type-specific antigen (matrix protein) and DBHM which identifies the serovariant of influenza virus.

[Analysis of the antigenic and immunogenic properties of protein M of the influenza virus by an immunoenzyme method]. / Analiz antigennykh i immunogennykh svoistv belka M u virusa grippa immunofermentnym metodom.

A test system permitting the detection of influenza virus protein M at a concentration of 0.1-0.5 ng/ml in ELISA has been developed. The use of this system made it possible to detect influenza viruses A and B directly in crude virus-containing material and clinical samples obtained from influenza patients. During the outbreak of influenza in the spring of 1983 ELISA was successfully used for the rapid diagnosis of influenza, and some of its advantages in comparison with the conventional immunofluorescence test were thus demonstrated. To overcome difficulties arising from the low immunogenic potency of protein M, in the process of obtaining diagnostic sera and ascitic fluids the animals were immunized with the conjugate of protein M and polyelectrolite, which ensured considerable activation of humoral immune response.

[Antigenic properties of the M1 proteins of influenza type B viruses and the detection of a common antigenic determinant of the M1 proteins of type A and B viruses]. / Antigennye svoistva belkov M1 virusov grippa tipa B i vyiavlenie obshchei antigennoi determinanty belkov M1 virusov tipa A i B.

Antigenic properties of M1 proteins of influenza B viruses were studied by indirect EIA using monospecific antisera to M1 protein obtained by immunization of rabbits and mice. Antisera with highest titres were obtained by immunization of mice with polyelectrolyte-conjugated M1 protein. Antigenic heterogeneity of M1 proteins of influenza B viruses was demonstrated. M1 proteins of B/Lee/40 and B/USSR/03/84 viruses reacted with B/Hong Kong/72 antiserum to one-fourth of the homologous titre. Cross-experiments using M1 proteins recovered from influenza type A and B viruses and antisera to them revealed heterologous reactions suggesting a common antigenic determinant in A and B M1 proteins. Treatment of the antisera with M1 protein of type A resulted in a decline of antibody titres to M1 proteins of type B. The two available monoclones to M1 protein of type A reacted only with M1 A but not with M1 B.

[Rapid diagnosis of influenza by detecting viral M protein in an immunoenzyme test]. / Ekspress-diagnostika grippa s pomoshch'iu vyiavleniia virusnogo belka M v immnofermentnom teste.

Previously developed ELISA test system with sensitivity about 0.5 ng/ml of virus protein M was used for influenza virus detection in nasopharyngeal washings of patients with acute respiratory virus diseases (ARVD). Altogether 184 specimens from patients with ARVD and 31 from patients with other infections were examined in the pre-epidemic and epidemic periods of 1983. In parallel, virus antigens were detected by direct immunofluorescence test (IF). The total frequency of viral antigen detection by ELISA and IF coincided (63% of the specimens examined). With the specimens collected early in the disease (2-3 days) the rate of virus findings by ELISA rose to 82%. Direct detection of viral antigen in nasopharyngeal washings by means of an objective immunochemical method seems to be promising for a large-scale rapid diagnosis of influenza.