Vol 67, No 6 (2022)

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Genetic diversity of Siberian bovine coronavirus isolates (Coronaviridae: Coronavirinae: Betacoronavirus-1: Bovine-Like coronaviruses)

Glotov A.G., Nefedchenko A.V., Yuzhakov A.G., Koteneva S.V., Glotova T.I., Komina A.K., Krasnikov N.Y.


Introduction. Bovine coronaviruses (BCoVs) are causative agents of diarrhea, respiratory diseases in calves and winter cow dysentery. The study of genetic diversity of these viruses is topical issue.

The purpose of the research is studying the genetic diversity of BCoV isolates circulating among dairy cattle in Siberia.

Materials and methods. Specimens used in this study were collected from animals that died or was forcedly slaughtered before the start of the study. The target for amplification were nucleotide sequences of S and N gene regions.

Results. Based on the results of RT-PCR testing, virus genome was present in 16.3% of samples from calves with diarrheal syndrome and in 9.9% with respiratory syndrome. The nucleotide sequences of S gene region were determined for 18 isolates, and N gene sequences - for 12 isolates. Based on S gene, isolates were divided into two clades each containing two subclades. First subclade of first clade (European line) included 11 isolates. Second one included classic strains Quebec and Mebus, strains from Europe, USA and Korea, but none of sequences from this study belonged to this subclade. 6 isolates belonged to first subclade of second clade (American-Asian line). Second subclade (mixed line) included one isolate. N gene sequences formed two clades, one of them included two subclades. First subclade included 3 isolates (American-Asian line), and second subclade (mixed) included one isolate. Second clade (mixed) included 8 sequences. No differences in phylogenetic grouping between intestinal and respiratory isolates, as well as according to their geographic origin were identified.

Conclusion. The studied population of BCoV isolates is heterogeneous. Nucleotide sequence analysis is a useful tool for studying molecular epidemiology of BCoV. It can be beneficial for choice of vaccines to be used in a particular geographic region.

Problems of Virology. 2022;67(6):465-474
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Variability of genes encoding nonstructural proteins of rotavirus А (Reoviridae: Rotavirus: Rotavirus A) genotype G9P[8] during the period of dominance in the territory of Nizhny Novgorod (central part of Russia) (2011–2020)

Velikzhanina E.I., Sashina T.A., Morozova O.V., Epifanova N.V., Novikova N.A.


Introduction. In Russia, rotavirus A is the main cause of severe viral gastroenteritis in young children. The molecular features that allow a rotavirus of a particular genotype to gain an evolutionary advantage remain unclear, therefore, the study of the genetic diversity of rotaviruses based on genes encoding nonstructural proteins (NSPs) responsible for the reproduction of the virus in the cell is an urgent task.

Objective. To study the genetic diversity of rotaviruses of genotype G9P[8], which dominated Nizhny Novgorod in 2011–2020, based on genes encoding nonstructural proteins.

Materials and methods. Rotavirus-positive samples were subjected to PCR-genotyping and sequencing of NSP1 NSP5 genes. Phylogenetic analysis was carried out in the MEGA X program.

Results. In the period 2011–2020, G9P[8] rotaviruses with four variants of the NSP2 gene were co-circulating in Nizhny Novgorod. New alleles were noted in 2012 (N1-a-III), 2016 (N1-a-IV) and in 2019 (N1-a-II). The appearance of new variants of other genes occurred in 2014 (E1-3, NSP4), 2018 (T1-a3-III, NSP3) and in 2019 (A1-b-II, NSP1). NSP2 gene had the most variable amino acid sequence (16 substitutions), 2 to 7 substitutions were observed in NSP1, NSP3 and NSP4, NSP5 was conservative.

Discussion. The results obtained are consistent with the literature data and indicate the participation of NSP genes in maintaining the heterogeneity of the rotavirus population.

Conclusion. Until 2018, the genetic diversity of rotaviruses in Nizhny Novgorod was determined by the circulation of strains carrying several alleles of the NSP2 gene and conservative genes NSP1, NSP3NSP5. By the end of the study period, new variants of the genotype G9P[8] were formed in the population, carrying previously unknown combinations of alleles of nonstructural genes.

Problems of Virology. 2022;67(6):475-486
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Molecular and genetic characteristics of the multicomponent flavi-like Kindia tick virus (Flaviviridae) found in ixodes ticks on the territory of the Republic of Guinea

Kartashov M.Y., Gladysheva A.V., Naidenova E.V., Zakharov K.S., Shvalov А.N., Krivosheina E.I., Senichkina A.M., Bah M.B., Ternovoi V.A., Boumbaly S., Loktev V.B.


Introduction. Ixodes ticks are vectors for pathogens of many infectious diseases. Recently, during the study of Rhipicephalus geigyi ticks collected from livestock in the Republic of Guinea, a new multicomponent flavi-like RNA virus, called Kindia tick virus (KITV), was discovered with an unusual mechanism for the implementation of genetic information.

The aim of the work is to detect and study the genetic diversity of KITV in ixodes ticks collected in the territory of the Kindia province of the Republic of Guinea.

Material and methods. In 2021, 324 specimens of ticks of the species Amblyomma variegatum, Rh. geigyi, Rh. annulatus, Rh. decoloratus, Rh. senegalensis were collected from cattle. The detection of viral RNA was carried out in individual samples of ticks by RT-PCR, followed by the determination of the nucleotide sequence and phylogenetic analysis.

Results and discussion. KITV detection rates in ticks of the species Rh. geigyi was 12.2%, Rh. annulatus – 4.4%, Rh. decoloratus – 3.3%. However, the KITV genetic material has not been identified in Am. variegatum ticks, which are one of the dominant species in West Africa. For all virus isolates, a partial nucleotide sequences of each of the four viral segments (GenBank, OK345271–OK345306) were determined. The phylogenetic analysis showed a high level of identity (98.5–99.8%) for each of the four segments of the viral genome with those previously found in the Republic of Guinea. The obtained KITV isolates are most genetically close to Mogiana tick virus, which was previously detected in South America in Rh. microplus ticks and significantly differed from other multicomponent viruses circulating in Europe and Asia, including the Russian Federation.

Conclusion. KITV genetic material was found in three species of ixodid ticks collected from livestock in a number of prefectures of the Republic of Guinea. The infection rate in ticks was 3.3–12.2%. The continuation of research in this direction remains relevant.

Problems of Virology. 2022;67(6):487-495
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Molecular epidemiological analysis of SARS-CoV-2 genovariants in Moscow and Moscow region

Ozhmegova E.N., Savochkina T.E., Prilipov A.G., Tikhomirov E..., Larichev V.F., Sayfullin M.A., Grebennikova T.V.


Introduction. SARS-CoV-2, a severe acute respiratory illness virus that emerged in China in late 2019, continues to spread rapidly around the world, accumulating mutations and thus causing serious concern. Five virus variants of concern are currently known: Alpha (lineage B.1.1.7), Beta (lineage B.1.351), Gamma (lineage P.1), Delta (lineage B.1.617.2), and Omicron (lineage B.1.1.529). In this study, we conducted a molecular epidemiological analysis of the most prevalent genovariants in Moscow and the region.

The aim of the study is to estimate the distribution of various variants of SARS-CoV-2 in Moscow city and the Moscow Region.

Materials and methods. 227 SARS-CoV-2 sequences were used for analysis. Isolation of the SARS-CoV-2 virus was performed on Vero E6 cell culture. Sequencing was performed by the Sanger method. Bioinformatic analysis was carried out using software packages: MAFFT, IQ-TREE v1.6.12, jModelTest 2.1.7, Nextstrain, Auspice v2.34.

Results. As a result of phylogenetic analysis, we have identified the main variants of the virus circulating in Russia that have been of concern throughout the existence of the pandemic, namely: variant B.1.1.7, which accounted for 30% (9/30), AY.122, which accounted for 16.7% (5/30), BA.1.1 with 20% (6/30) and B.1.1 with 33.3% (10/30). When examining Moscow samples for the presence of mutations in SARS-CoV-2 structural proteins of different genovariants, a significant percentage of the most common substitutions was recorded: S protein – D614G (86.7%), P681H/R (63.3%), E protein – T9I (20.0%); M protein – I82T (30.0%), D3G (20.0%), Q19E (20.0%) and finally N protein – R203K/M (90.0%), G204R/P (73.3 %).

Conclusion. The study of the frequency and impact of mutations, as well as the analysis of the predominant variants of the virus are important for the development and improvement of vaccines for the prevention of COVID-19. Therefore, ongoing molecular epidemiological studies are needed, as these data provide important information about changes in the genome of circulating SARS-CoV-2 variants.

Problems of Virology. 2022;67(6):496-505
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Antiviral and virucidal activity of sodium deoxyribonucleate and its complex with iron against viruses of different kingdoms and families

Nosik D.N., Kalnina L.B., Lobach O.A., Chataeva M.S., Berezhnaya E.V., Bochkova M.S., Kiseleva I.A., Selimova L.M., Nosik N.N.


Introduction. The urgent problem of modern medicine is the fight against acute respiratory viral infections (ARVI). To combat ARVI, drugs of wide antiviral potency are needed, as well as immunomodulating drugs. Such antiviral and immunomodulatory effects has sodium deoxyribonucleate (DNA-Na) and its complex with iron (DNA-Na-Fe) developed on the basis of double-stranded DNA of natural origin.

Aim of the study: To assess antiviral and virucidal activity of DNA-Na and DNA-Na-Fe against viruses of different kingdoms and families.

Materials and methods. Antiviral and virucidal activity of DNA-Na and DNA-Na-Fe was assessed in cell cultures infected with viruses.

Results and discussion. DNA-Na and DNA-Na-Fe had antiviral activity against adenovirus at concentrations of 250–1000 mcg/ml. Antiviral effect of both drugs was not detected in case of poliovirus. DNA-Na and DNA-Na-Fe had antiviral activity against coronavirus in all administration schemes. EC50 for DNA-Na ~ 2500 mcg/ml, for DNA-Na-Fe ~ 1000 mcg/ml. In cells treated with DNA-Na-Fe, secretion of following pro–inflammatory cytokines was detected: Interleukin (IL) 1β, IL-2, IL-6, IL-18, interferon-α (IFN-α), IFN-γ, as well as anti-inflammatory cytokines: IL-4, IL-10, antagonist of IL-1 receptor. Evidently, DNA-Na and DNA-Na-Fe have antiviral effect, but mechanism of action does not seem to be associated with specific effect on viral replication. Presence of virucidal activity of drugs against representatives of Coronaviridae, Adenoviridae, Picornaviridae, Retroviridae, Herpesviridae in vitro test in range of 1.0–3.0 lg TCID50 was identified.

Conclusion. Presence of simultaneous antiviral and virucidal activity of DNA-Na and DNA-Na-Fe against adeno- and coronaviruses shows their prospects for prevention and treatment of ARVI.

Problems of Virology. 2022;67(6):506-515
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Adjuvant effect of dispersed fullerene C60 on the immune response to constructs harboring amino acid and nucleotide sequences of hepatitis C virus nonstructural NS5B protein

Masalova O.V., Lesnova E.I., Andreev S.M., Shershakova N.N., Kozlov V.V., Permyakova K.Y., Demidova N.A., Valuev-Elliston V.T., Turetskiy E.A., Ivanov A.V., Nikolaeva T.N., Khaitov M.R., Pronin A.V., Kushch A.A.


Introduction. A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a weak immune response and require the use of adjuvants.

The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid.

Materials and methods. An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B – intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated.

Results. Mice immunization with rNS5B in a mixture with dnC60 at doses of 2–50 µg/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 7–20 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60.

Conclusions. Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.

Problems of Virology. 2022;67(6):516-526
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Assesment of specific T-cell immunity to SARS-CoV-2 virus antigens in COVID-19 reconvalescents

Blyakher M.S., Fedorova I.M., Tulskaya E. ., Kapustin I.V., Koteleva S.I., Ramazanova Z.K., Odintsov E.E., Sandalova S.V., Novikova L.I.


Introduction. The development of the COVID-19 pandemic has stimulated the scientific research aimed at studying of the mechanisms of formation the immunity against SARS-CoV-2. Currently, there is a need to develop a domestic simple and cost-effective specific method suitable for monitoring of T-cell response against SARS-CoV-2 in reconvalescents and vaccinated individuals.

Aim: Development of a screening method for evaluation specific T-cell immunity against SARS-CoV-2.

Materials and methods. Total 40 individuals who had mild to moderate COVID-19 and 20 healthy volunteers who did not have a history of this disease were examined. The presence and levels of IgG and IgM antibodies to SARS-CoV-2 were identified in participant’s sera by ELISA using the diagnostic kits from JSC “Vector-Best” (Novosibirsk, Russian Federation). Antigenic stimulation of mononuclear cells was carried out on commercial plates with adsorbed whole-virion inactivated SARS-CoV-2 antigen (State Research Center of Virology and Biotechnology VECTOR Novosibirsk, Russian Federation). The concentration of IFN-γ was measured in ELISA using the test systems from JSC “Vector-Best” (Novosibirsk, Russian Federation). The immunophenotyping of lymphocytes was performed on a flow cytometer Cytomics FC500 (Beckman Coulter, USA). Statistical data processing was carried out using the Microsoft Excel and STATISTICA 10 software package.

Results. Stimulation of mononuclear cells isolated from the peripheral blood with whole-virion inactivated SARS-CoV-2 antigen fixed at the bottom of the wells of a polystyrene plate showed a significantly higher median response in terms of IFN-γ production in 40 people who had history of COVID-19 compared to 20 healthy blood donors (172.1 [34.3–575.1] pg/ml versus 15.4 [6.9–25.8] pg/ml, p < 0.0001).

There was no difference in median IFN-γ levels in supernatants collected from unstimulated mononuclear cells from COVID-19 reconvalescents and healthy donors (2.7 [0.4–11.4] pg/ml versus 0.8 [0.0–23.3] pg/ml, p < 0.05). The overall sensitivity and specificity of this method were 73% (95% CI 58–88%) and 100% (95% CI 100–100%), respectively, at a cut-off of 50 pg/ml.

Conclusion. The developed method for assessment of the cellular immune response to SARS-CoV-2 can be used as a screening method for monitoring the T-cell response in a population against a new coronavirus infection in recovered people.

Problems of Virology. 2022;67(6):527-537
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In No. 5, 2022 of the journal "Questions of Virology" in the section "Editorial concept" the article "130 years of virology" was published (Lvov D.K., Alkhovsky S.V., Zhirnov O.P. 130 years of virology. Questions of virology. 2022; 67(5): 357-384. DAY: https://doi.org/10.36233/0507-4088-140).
The review presents the main stages of the formation and development of virology as a science in Russia with an emphasis on the most significant achievements of domestic virologists in the fight against viral infectious diseases of humans and animals
The editorial office received the following reviews and reviews of the article.

Problems of Virology. 2022;67(6):538-540
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