Genome stability of the vaccine strain VACΔ6.

Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against orthopoxvirus infections has virtually been lost. Therefore, the risk of spreading zoonotic human orthopoxvirus infections caused by monkeypox and cowpox viruses has increased in the world. First-generation smallpox vaccines based on Vaccinia virus (VAC) are reactogenic and therefore not suitable for mass vaccination under current conditions. This necessitates the development of modern safe live vaccines based on VAC using genetic engineering. We created the VACΔ6 strain by transient dominant selection. In the VACΔ6 genome, f ive virulence genes were intentionally deleted, and one gene was inactivated by inserting a synthetic DNA fragment. The virus was passaged 71 times in CV-1 cells to obtain the VACΔ6 strain from the VAC LIVP clonal variant. Such a long passage history might have led to additional off-target mutations in VACΔ6 compared to the original LIVP variant. To prevent this, we performed a genome-wide sequencing of VAC LIVP, VACΔ6, and f ive intermediate viral strains to assess possible off-target mutations. A comparative analysis of complete viral genomes showed that, in addition to target mutations, only two nucleotide substitutions occurred spontaneously when obtaining VACΔ4 from the VACΔ3 strain; the mutations persisting in the VACΔ5 and VACΔ6 genomes. Both nucleotide substitutions are located in intergenic regions (positions 1431 and 189738 relative to LIVP), which indicates an extremely rare occurrence of off-target mutations when using transient dominant selection to obtain recombinant VAC variants with multiple insertions/deletions. To assess the genome stability of the resulting attenuated vaccine strain, 15 consecutive cycles of cultivation of the industrial VACΔ6 strain were performed in 4647 cells certif ied for vaccine production in accordance with the "Guidelines for Clinical Trials of Medicinal Products". PCR and sequencing analysis of six DNA fragments corresponding to the regions of disrupted genes in VACΔ6 showed that all viral DNA sequences remained unchanged after 15 passages in 4647 cells.

Increased LAMP1 Expression Enhances SARS-CoV-1 and SARS-CoV-2 Production in Vero-Derived Transgenic Cell Lines.

Coronaviridae is a family of single-stranded RNA (ssRNA) viruses that can cause diseases with high mortality rates. SARS-CoV-1 and MERS-CoV appeared in 2002‒2003 and 2012, respectively. A novel coronavirus, SARS-CoV-2, emerged in 2019 in Wuhan (China) and has caused more than 5 million deaths in worldwide. The entry of SARS-CoV-1 into the cell is due to the interaction of the viral spike (S) protein and the cell protein, angiotensin-converting enzyme 2 (ACE2). After infection, virus assembly occurs in Golgi apparatus-derived vesicles during exocytosis. One of the possible participants in this process is LAMP1 protein. We established transgenic Vero cell lines with increased expression of human LAMP1 gene and evaluated SARS-CoV-1 and SARS-CoV-2 production. An increase in the production of both viruses in LAMP1-expressing cells when compared with Vero cells was observed, especially in the presence of trypsin during infection. From these results it can be assumed that LAMP1 promotes SARS-CoV-1 and SARS-CoV-2 production due to enhanced exocytosis.

[Increased LAMP1 Expression Enhances SARS-CoV-1 and SARS-CoV-2 Production in Vero-Derived Transgenic Cell Lines].

Coronaviridae is a family of single-stranded RNA (ssRNA) viruses that can cause diseases with high mortality rates. SARS-CoV-1 and MERS-CoV appeared in 2002-2003 and 2012, respectively. A novel coronavirus, SARS-CoV-2, emerged in 2019 in Wuhan (China) and has caused more than 5 million deaths in worldwide. The entry of SARS-CoV-1 into the cell is due to the interaction of the viral spike (S) protein and the cell protein, angiotensin-converting enzyme 2 (ACE2). After infection, virus assembly occurs in Golgi apparatus-derived vesicles during exocytosis. One of the possible participants in this process is LAMP1 protein. We established transgenic Vero cell lines with increased expression of human LAMP1 gene and evaluated SARS-CoV-1 and SARS-CoV-2 production. An increase in the production of both viruses in LAMP1-expressing cells when compared with Vero cells was observed, especially in the presence of trypsin during infection. From these results it can be assumed that LAMP1 promotes SARS-CoV-1 and SARS-CoV-2 production due to enhanced exocytosis.

Detection of SARS-CoV-2 RNA in nasopharyngeal swabs from COVID-19 patients and asymptomatic cases of infection by real-time and digital PCR.

In this work we tested two reagent kits developed by us for detecting SARS-CoV-2 RNA using a fragment of the ORF1ab gene in digital PCR and real-time PCR formats. Data were obtained on the detection of SARS-CoV-2 virus RNA in nasopharyngeal swabs of patients with COVID-19 and asymptomatic carriers. The developed reagent kits provided 100% sensitivity and a detection limit of 103 GE / ml for qPCR, and at least 200 copies / ml of viral RNA when performing digital PCR. These methods were tested using a panel of 1,328 samples collected from patients with suspected COVID-19 at the beginning of 2020 in the Russian Federation. It has been shown that dPCR is more sensitive and can be used to analyze samples with low viral load, including those from patients without clinical symptoms. dPCR significantly improves the accuracy of laboratory research and significantly reduces the number of false negative results in the diagnosis of SARS-CoV-2. Determination of the concentration of SARS-CoV-2 RNA in patients with different clinical course of the disease showed that the concentration of viral RNA can sharply decrease in the first days of the disease. A low concentration of viral RNA in samples from patients is also characteristic of asymptomatic disease. Digital PCR provides a higher detection rate for asymptomatic cases, which is approximately 75% of those infected, as opposed to 45% for real-time PCR. The results obtained on the use of the digital PCR method for detecting SARS-CoV-2 RNA showed that this method is especially suitable for detecting RNA in case of its low concentration in contacts, as well as for monitoring changes in viral load in convalescent patients.

[Development of the drug oncolytic immunotherapy based on vaccinia viruses (Vaccinia virus, Orthopoxvirus, Chordopoxvirinae, Poxviridae) against breast cancer.]

INTRODUCTION: Currently, new directions in cancer therapy are actively developing, one of which is oncolytic immunotherapy. This approach would be to use of viruses as cancer specific cytolytic agents capable of stimulating both the tumor-specific and non-specific immune response. The objective paper was obtain a recombinant vaccinia virus containing genes encoding immunostimulating molecules and study oncolytic and immunostimulating properties of recombinant virus. MATERIAL AND METHODS: MTT test, ELISA, methods of transient dominant selection. RESULTS: The recombinant vaccinia virus (L-IVP_oncoB) were obtained with deletion of the gene encoding thymidine kinase and had an integrated gene encoding GM-CSF. Also the virus have deletion of the gene encoding viral growth factor and integrated genes encoding synthetic tumor-specific polyepitopic immunogens. It was shown that the modifications made to the viral genome did not affect the growth characteristics of the virus when cultured on CV-1 and 4647 cell cultures, and the cytopathogenic efficacy of the virus was determined in relation to cancer cultures of cells of various genesis. In in vivo experiment, it was revealed that the polyepitopic construct in the genome L-IVP_oncoB is able to initiate a change in the profile of cytokines. DISCUSSION: The obtained data characterized L-IVP_oncoB as a promising cytopathogenic and immunostimulating agent and showed the need for further study of its properties as means of oncolytic immunotherapy. CONCLUSION: The basic experiments on the evaluation of the biological properties of the obtained L-IVP_oncoB, which are necessary for the characterization of the oncolytic virus, have been carried out.

An Outbreak of Sheep Pox in Zabajkalskij kray of Russia.

In this study, we investigated recent sheep pox outbreaks that occurred in Ononsky and Borzunsky regions of Zabajkalskij kray of Russia. The outbreaks involved in 2756 animals of which 112 were infected and 3 were slaughtered. Samples of injured skin of infected sheep were analysed by electron microscopy and CaPV-specific P32 gene amplification. Following sequence analysis of entire P32 gene showed that both specimens were identical to the sequence of several sheep poxvirus isolates from China and India. The close location of China to the last decade's Russian outbreaks suggest that possible future outbreaks in Russia could occur along the border regions with countries where sheep and goat pox are not controlled.

[The development of method of detection of human pathogenic orthopoxviruses on the basis of polymerase chain reaction in real time].

The set of specific oligonucleotide primers and hybridization testers with the support of TaqMan in real time is proposed to identify and differentiate such human pathogenic orthopoxviruses as smallpox virus, monkey smallpox virus, cowpox virus, variolovaccine. The analytical specificity of the set is determined upon the example of 20 strains of 6 types of human pathogenic orthopoxviruses and made up 100%. The sensibility is established using recombinant plasmids containing orthopoxviruses sequence. The minimal revealed amount of plasmid made up to 20 copies for smallpox and monkey smallpox viruses, 10 copies for cowpox and 60 copies for variolovaccine in reaction.

[Development of current smallpox vaccines].

The review gives data on the history of smallpox vaccination and shows the high topicality of designing the current safe vaccines against orthopoxviruses. Four generations of live smallpox, protein subunit, and DNA vaccines are considered. Analysis of the data published leads to the conclusion that it is promising to use the up-to-date generations of safe smallpox subunit or DNA vaccines for mass primary immunization with possible further revaccination with classical live vaccine.

[Phytoplankton in reservoir R-11 of Techa reservoir cascade of Industrial Association "Maiak"].

Phytoplankton of reservoir R-11 was investigated at vegetative seasons of 2007 and 2008. R-11 is a locking reservoir in the cascade of Mayak PA reservoirs. The specific activity in water of R-11 ranged from 0.9 to 1.8 kBq/dm for 90Sr and from 0.98 to 3.97 kBq/dm3 for 137Cs; in bottom sediments it ranged from 20 to 400 kBq/kg for 90Sr and from 0.35 to 220 kBq/kg for 137Cs. Concentration of SO4- exceeded the maximum permissible concentration for fishing reservoirs in 5-7 times. The content of 90Sr in the phytoplankton has made 400 kBq/kg (for dry weight), and 137Cs--2.20 kBq/kg (for dry weight). The absorbed dose of irradiation for the phytoplankton calculated under the content of 137Cs and 90Sr in water and in phytoplankton (the external irradiation from activity of bottom sediments was not considered) has made 901 mGy per year. Altogether the phytoplankton of reservoir R-11 included 107 species. Such species of Cyanobacteriae as Oscillatoria agardhii Gom. was the dominant in phytoplanktonic community. Another Cyanobacteriae, Lyngbya limnetica Lemm. and Aphanizomenonflos-aquae (L.) Ralfs. acted the most important part in a complex of species-subdominants. According to quantitative development of the phytoplankton the reservoir R-11 can be classified as P-mezosaprobe waters.

[Identification of human pathogenic variola and monkeypox viruses by real-time polymerase chain reaction].

A kit of specific oligonucleotide primers and hybridization probes has been proposed to detect orthopoxviruses (OPV) and to discriminate human pathogenic viruses, such as variola virus and monkey virus by real-time polymerase chain reaction (PCR). For real-time PCR, the following pairs of fluorophore and a fluorescence quencher were used: TAMRA-BHQ2 for genus-specific probes and FAM-BHQ1 for species-specific ones (variola virus, monkeypox virus, ectomelia virus). The specificity of this assay was tested on 38 strains of 6 OPV species and it was 100%.

[Modifications in major satellite methylation in the nucleus of a two-cell mouse embryo dependent on developmental conditions].

The study of the degree of DNA methylation in the nucleus, in particular of the major satellite in two-cell mouse embryos developing in the maternal organism, in standard cultural media M16 used for cultivation of mouse embryos and M2 media used for manipulations with embryos in the air was conducted. Two-cell embryos nucleus aged 44-46 hours after chorionic hormone injection were investigated. The revealed results are evidence for the dependence of the major satellite Ts methylation level of the developmental conditions of embryos. The methylation level of the nucleus DNA was shown to increase with a deterioration of environmental conditions. It was reported, that in the case of cultivation in M2 media not suitable for long cultivation, the DNA Ts methylation level, MaSat in particular, was higher compared to other embryo groups. Accordingly, not only a significant number of genes but also repeats of satellite DNA are involved in epigenetic regulation.

[Localization of satellite DNA and associated protein in respect to nucleolar precursor bodies in one- and two-cell mouse embryos].

Nucleolar precursor bodies (NPB) are characteristic structures in the nuclei of one- and two cell mouse embryos. The alignment of centromeric (CEN) and pericentromeric (periCEN) chromosome regions to the chromatin layer surrounding NPB is known. Mus musculus 4 satellite DNA (satDNA) types are known to be located in CEN region--mouse minor satellite (MiSat) and mouse satellite 3 (MS3); and periCEN region--mouse major satellite (MaSat) and mouse satellite (MS4). We determined the localization of 4 types of mouse satDNA CEN and periCEN regions and associated proteins: RNA-helicase p68, SMC3, Rad21 subunits of the cohesin complex and SYCP3 subunit of the synaptonemal complex (SC). Partially flattened nuclei of the one- and two-cell embryos and embryos treated with ocadaic acids (OA) were used. Different satDNA fragments revealed distinct domains at the surface of NPB: periCEN MaSat was always localized in NPB more internally covering almost entire surface of NPB while CEN MiSat, MS3 and periCEN MS4 showed more peripheral localization. All 4 satDNA did not cover the entire areas of the NPB, indicating the presence of other DNA sequence involved in its formation. RNA-helicase p68 and components of multiprotein cohesin and synaptonemal complexes are the necessary components of NPB. Our results support the opinion that NPB serve as a precursor of chromocenters.

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum.

Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria.

Multiplex PCR detection and species differentiation of orthopoxviruses pathogenic to humans.

A method for one-stage rapid identification of four orthopoxvirus species pathogenic to humans based on multiplex polymerase chain reaction (MPCR) was developed. Five pairs of oligonucleotide primers--one, genus-specific; and the rest, species-specific for variola, monkeypox, cowpox, and vaccinia viruses, respectively--were used concurrently for MPCR assay of orthopoxvirus DNAs. Specificity and sensitivity of the method developed were evaluated using DNAs of 57 orthopoxvirus strains, including the DNAs isolated from human case clinical materials.

[Multiplex PCR analysis for species specific express-identification of orthopoxviruses]. / Mul'tipleksnyi PTsR-analiz dlia vidospetsifichnoi ékspress-identifikatsii ortopoksvirusov.

A method for one-stage rapid identification of the four orthopovirus species, which are pathogenic to humans, involving multiplex polymerase chain reaction (MPCR) was developed. Five pairs of oligonucleotides primers were simultaneously used in the mentioned MPCR assay of orthopoxvirus DNA; one of the pairs was genus-specific, the remaining four primers were species-specific for variola, monkey-pox, cowpox and vaccine-pox. The specificity and sensitivity of the developed method were evaluated through analyzing the DNA samples of 55 orthopoxvirus strains, including samples isolated from human clinical materials.

Adenosine as a Modulator of Functional Activity of Peripheral Blood Phagocytes in Asthmatic Patients and Healthy Volunteers.

The influence of adenosine (1.0 &mgr;M) on generation of reactive oxygen species and myeloperoxidase activity of adherent phagocytes from healthy donors and asthmatic patients was studied in vitro. Adenosine enhanced NADP-oxidase activity and inhibited myeloperoxidase in leukocytes from healthy donors. It did not influence NADP-oxidase activity but stimulated myeloperoxidase activity in atopic asthma patients. In non-atopic asthma the activity of the both enzymes was inhibited by adenosine. The effects of adenosine depended on the stage and duration of asthma. The hypothesis was proposed that in bronchial asthma adenosine should play a protective role against tissue damage produced by activated phagocytes.

[The work capacity expertise of motor vehicle drivers suffering from IHD with rhythm disorders and the possibilities for their social and vocational rehabilitation]. / Ekspertiza trudosposobnosti voditelei avtotransporta, stradaiushchikh IBS s narusheniiami ritma, i vozmozhnosti ikh sotsial'no-trudovoi reabilitatsii.

The paper presents the results of clinical and labour examinations of 51 drivers with cardiac arrhythmia. Long-term postinfarctional ECG monitoring was attempted in 30 drivers in the course of driving. The frequency of arrhythmia episodes correlated with the presence of extrasystole in acute and subacute period of the disease, anginal functional class, the size and time of myocardial infarction. Single episodes of ventricular extrasystole registered in the course of driving did not interfere with professional activities because such extrasystoles did not imply the risk of more severe arrhythmia. Labour prognosis in drivers with frequent, polytopic ventricular extrasystoles or fibrillation was much worse.

[Bacillus subtilis gene rec223: molecular cloning and proposed function of its protein product]. / Gen rec223 Bacillus subtilis: molekuliarnoe klonirovanie i predpolagaemye funktsii ego belkovogo produkta.

Chromosomal DNA fragment which complemented rec223 mutation of Bacillus subtilis was cloned. Introduction of one copy of the cloned gene into the cells of the rec mutant restored both normal activity for DNA damages repair after mitomycin C action and recombination proficiency. Using multicopy vector led to no formation of recombinants, which was probably connected with overproduction of rec223 gene protein product in Bacillus subtilis cells.