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Introduction: Rotavirus infection is the major cause of severe dehydrating diarrhea requiring hospitalization in young children worldwide. Due to their segmented genome, rotaviruses are capable of gene reassortment, which makes the emergence and spread of genetically novel strains possible. The purpose of this study was to search for unusual rotaviruses circulating in Nizhny Novgorod in 2021‒2023 and their molecular genetic characterization based on all genome segments., Materials and Methods: Rotavirus-positive stool samples of children were examined by PCR genotyping and electrophoresis in PAAG. cDNA fragments of each of the 11 genes ( VP1‒VP4 , VP6 , VP7 , NSP1‒NSP5 ), 570 to 850 nucleotide pairs in length were sequenced for the selected strains. The phylogenetic analysis was performed in the MEGA X program., Results: In the study period 2021‒2023, 11 G[P] combinations with a predominance of G3P[8] (59.5%) were identified. Six atypical Rotavirus А (RVA) strains were identified: 2 strains of the G2P[4] genotype (G2-P[4]-I2-R2-C2-M2-A3-N2-T3-E2-H3, G2-P[4]-I2-R2-C2-M2-A3-N2-T3-E3-H2) and 4 G3P[9] strains (all strains had the genotype G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3). Phylogenetic analysis based on all genes showed an evolutionary relationship between rotaviruses similar to rotaviruses of cats and dogs (BA222-like) and unusual strains of the G2P[4] genotype, for which a mixed combination of genotypes was identified and characterized for the first time., Discussion: The results obtained expand the understanding of the diversity of reassortant RVAs, as well as complement the data on the genotypic structure of the rotavirus population in Nizhny Novgorod., Conclusion: The wide genetic diversity of reassortant RVA can help rotaviruses overcome the immunological pressure provided by natural and vaccine-induced immunity. In this regard, to control the emergence of new variants and assess changes in the virulence of rotaviruses after reassortment processes, continuous molecular monitoring for circulating RVA is necessary.

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 20112020, 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 20112020, 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.

Introduction: The novel coronavirus infection COVID-19 is a major public health problem worldwide. Several publications show the presence of gastrointestinal (GI) symptoms (nausea, vomiting, and diarrhea) in addition to respiratory disorders.The aim of this study was the monitoring of RNA of COVID-19 pathogen, coronavirus SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) in children hospitalized with acute intestinal infection (AII), with following molecular-genetic characterization of detected strains., Material and Methods: Fecal samples of children with AII hospitalized in infectious hospital of Nizhny Novgorod (Russia) in the period from 01.07.2020 to 31.10.2021 were used as material for the study. Viral RNA detection was performed by real-time polymerase chain reaction (RT-PCR). The nucleotide sequence of S-protein gene fragment was determined by Sanger sequencing., Results and Discussion: SARS-CoV-2 genetic material was detected in 45 out of 2476 fecal samples. The maximum number of samples containing RNA of the virus occurred in November 2020 (detection rate of 12.2%). In 20.0% of cases, SARS-CoV-2 RNA was detected in combination with rota-, noro-, and adenoviruses. 28 nucleotide sequences of S-protein gene fragment complementary DNA (cDNA) were determined. Phylogenetic analysis showed that the studied SARS-CoV-2 strains belonged to two variants. Analysis of the S-protein amino acid sequence of the strains studied showed the absence of the N501Y mutation in the 2020 samples, which is a marker for variants with a high epidemic potential, called variants of concern (VOC) according to the World Health Organization (WHO) definition (lines Alpha B.1.1.7, Beta B.1.351, Gamma P.1). Delta line variant B.1.617.2 was identified in two samples isolated in September 2021., Conclusion: The detection of SARS-CoV-2 RNA in the fecal samples of children with AII, suggesting that the fecal-oral mechanism of pathogen transmission may exist, determines the necessity to optimize its monitoring and to develop an algorithm of actions with patients with signs of AII under the conditions of a novel coronavirus infection pandemic.

Introduction: The pentavalent rotavirus vaccine has been registered in Russia, however, the vaccination coverage remains low, and an annual increase in the incidence of rotavirus infection is unavoidable. In this regard, molecular monitoring of rotaviruses in order to search for new variants possessing epidemic potential is an urgent task., Material and Methods: PCR genotyping and VP4 and VP7 genes sequencing were used to characterize rotaviruses circulating in Nizhny Novgorod in 2012-2020. The phylogenetic analysis of the strains was carried out using the BEAST software package., Results: The spectrum included 17 genotypes with predominance of G9P[8] (37,4%). Detected in this study genotypes G1P[4], G1P[9], G2P[8], G4P[4], G4P[6], G8P[8], and G9P[4] were not previously identified in Nizhny Novgorod. The circulation of DS-1-like strains possessing genotypes G1P[8], G3P[8], G8P[8], or G9P[8] and a short RNA pattern had been shown. Rotaviruses of the common genotypes were genetically heterogeneous and belonged to different phylogenetic lineages and/or sublineages (P[4]-IV-a; P[4]-IV-b; P[8]-3.1; P[8]-3.3; P[8]-3.4 and P[8]-3.6; G1-I; G1-II; G2-IVa-1; G2-IVa-3; G3-1; G3-3; G4-I-c; G9-III; G9-VI)., Discussion: These results extend the available data on the genotypic structure of rotavirus populations in Russia and show the genetic diversity of viral strains. G3P[8] DS-1-like viruses were representatives of the G3-1 lineage, new for the territory of Russia, and had the largest number of amino acid substitutions in the VP7 antigenic epitopes., Conclusion: The emergence and spread of strains with new genetic features may allow rotavirus to overcome the immunological pressure formed by natural and vaccine-induced immunity, and maintain or increase the incidence of rotavirus infection.

Introduction: Rotavirus A is one of the leading causes of acute gastroenteritis in children in the first years of life. Rotavirus infection is currently classified as a preventable infection. The most abundant rotavirion protein is VP6., Material and Methods: Phylogenetic analysis and calculation of phylodynamic characteristics were carried out for 262 nucleotide sequences of the VP6 gene of rotavirus species A, isolated in Russia, using the BEAST v.1.10.4 software package. The derivation and analysis of amino acid sequences was performed using the MEGAX program., Results: This study provides phylodynamic characteristics of the rotaviruses in Russia based on the sequences coding VP6 protein. Bayesian analysis showed the circulation of rotaviruses of three sublineages of genotype I1 and three sublineages of genotype I2 in Russia. The level of accumulation of mutations was established, which turned out to be similar for genotypes I1 and I2 and amounted to 7.732E-4 and 1.008E-3 nucleotides/site/year, respectively. The effective population sizes based on nucleotide sequences of the VP6 I1 and I2 genotypes are relatively stable while after the 2000s there is a tendency of its decreasing. Comparative analysis of the amino acid sequences in the region of the intracellular neutralization sites A (231-260 aa) and B (265-292 aa) made it possible to reveal a mutation in position V252I in a proportion of Russian strains of genotype I1 some strains of genotypes I1 and I2 had mutation I281V. These substitutions were not associated with any sublineages to which the strains belong. The analysis of three T-cell epitopes revealed four amino acid differences (in aa positions 305, 315, 342, 348) that were associated with the first or second genogroup., Conclusion: Based on the phylodynamic characteristics and amino acid composition of antigenic determinants, it was concluded that the VP6 protein is highly stable and could potentially be a good model for development of a rotavirus vaccine.

Introduction: In recent years the presence of reassortant rotavirus strains is increasingly mentioned in the world due to the application of the full-genome based classification system. Information on the circulation of such strains in the territory of Russia is limited. The aim of this work was the development of the approach for determination of genotypes of segments encoding VP6 (I) and NSP4 (E) to reveal reassortant strains., Material and Methods: Rotavirus-positive samples were studied by means of nucleotide sequencing and multiplex PCR. Phylogenetic analysis was conducted using the Bayesian approach., Results: Three alleles of the VP6 gene (I1-1, I2-IV, I2-VII) and seven alleles of the NSP4 gene (E1-I, E1-III, E2-VI, E2-VII, E2-X, E2-XII, E3) were detected on the base of nucleotide sequences of Nizhny Novgorod rotaviruses. Taking into account these results, the oligonucleotide primers specific to genotypes I1, I2, I3 and E1, E2, E3 were designed. Optimal conditions for multiplex PCR were chosen. The method was tested using the strains collected in Nizhny Novgorod in 2018. The diversity of I and E genotypes was determined and various combinations with G and P genotypes were identified., Discussion: G9-P[8]-I1-E1 rotaviruses were predominant (32.7 %) and G2-P[4]-I2-E2 rotaviruses were in second place (29.1 %). Strains with genotypes G4-P[8]-I1-E2, G3-P[8]-I2-E2 and G2-P[4]-I2-E1 were detected sporadically. They had genes of two rotavirus genogroups, so can be considered to be reassortant., Conclusion: The proposed approach is a useful tool for the characterization of rotaviruses in the conditions of the beginning of vaccination against rotavirus infection in Russia., Competing Interests: The authors declare no conflict of interest.

Group A rotaviruses (RVA) are the main cause of viral gastroenteritis in children worldwide. In this study we provide the molecular characteristics of reassortant DS-1-like G1P[8] RVA strains detected in Russia for the first time. Previously, such reassortant strains were detected in Japan and Thailand. The G1P[8] RVAs with DS-1-like short electropherotype RNA-PAGE were isolated from children hospitalised with an acute gastroenteritis during the 2013-2014 period. The DS-1-like G1P[8] strains accounted for 2.6% of all RVA strains detected continuously throughout the season. A phylogenetic analysis was made on the basis of the established nucleotide sequences of genes VP7, VP8* (VP4), VP6 and NSP4. The Nizhny Novgorod strains belong to G1-I and G1-II alleles of VP7 gene and to P[8]-3 allele of VP4. According to their VP6 sequences, two Russian samples clustered with the reassortant strains isolated in Japan, Thailand and Australia and two other strains were phylogenetically close to the typical G2P[4] DS-1-like RVA. Nucleotide sequences of G1P[8] strains that belong to NSP4 gene form a separate cluster from G3P[8] DS-1-like rotaviruses detected in Thailand and Australia. The RVA alleles included in Rotarix and RotaTeq vaccine strains were clustered separately from the studied reassortant RVAs. On the grounds of phylogenetic analysis we assume a polyphyletic origin of reassortants between Wa- and DS-1-like strains. Mutation rates evaluated by Bayesian inference in clusters with reassortant RVA strains were 1.004Е-3 (VP7), 1.227E-3 (VP4), 3.909E-4 (VP6), and 4.014Е-4 (NSP4). Analysis of tMRCA showed relatively contemporary origin of alleles DS-1-like G1P[8] rotaviruses: VP7 - 1998 (G1-I) and 1981 (G1-II), VP4 - 1998, VP6 - 1994, NSP4 - 1979.

Since spontaneous mutagenesis and quasi-species rearrangements of the RNA-containing viruses, as well as an absence of both viral and cellular RNA reparation systems, causes resistance to originally effective antiviral drugs, combination therapy with nucleoside and non-nucleoside inhibitors of the viral enzymes in combination with immunomodulators is recommended. The use of specific immunoglobulins does not result in complete elimination of the flaviviruses but rather in possible antibody-dependent enhancement of the flavivirus infection by means of increased penetration of complexes of virions with specific antibodies into cells with receptors for Fc-fragments of immunoglobulins.

A total of 5230 specimens from children with gastroenteritis collected in Nizhny Novgorod in 2006-2010 were screened for human parechoviruses (HPeV). HPeV were observed every year with mean frequency of 6.16%. The majority of HpeV (65.83%) was detected in children younger than 3 years. The typing of 71 detected HPeV with the use of partial sequencing of the VP3-VP1 region revealed the presence of HPeV1 (91.55%), HpeV6 (5.63%), HPeV3 (3.08%), HPeV4 (1.54%). HPeV1B was predominant among HPeV1, HPeV1A was identified rarely. Six stains of HPEV1 formed separate phylogenetic cluster, had sequence gomology with HPEV1A or HPeV1B not more than 88% and could be characterized as members of a separate genotype HPeV1.

The nuclear polyhedrosis virus (NPV) DNA was detected in samples from eggs and caterpillars of the gypsy moth collected in natural populations of the Western Siberia and Ural by means of PCR with primers corresponding to the polyhedrin gene. According to censuring data, the gypsy moth populations of Western Siberia were at the depression stage. The NPV DNA detection frequencies in eggs (8.6 +/- 4.8% - 13.6 +/- 5.2%) and caterpillars (21.0 +/- 6.3% - 22.2 +/- 6.7%) were not significantly differed. In the Urals, collection of the insects was performed in their gradation focus at the phase of maximal abundance. The DNA detection rate in eggs (11.4 +/- 5.0%) was confidently (p < 0.001) lower than in caterpillars (59.8 +/- 5.6%). Consequently, variations of the NPV infection prevalence during ontogenesis of Lymantria dispar (L.) was associated with the gradation cycle of the insect population dynamics.

The currently used tick-borne encephalitis virus vaccines are based on the inactivation of tick-borne encephalitis virus (TBEV) of Far Eastern or West European genetic types from the primary cultures of chick embryo fibroblasts. Since the WHO recommends that vaccines should be designed using continuous cell cultures rather than chick embryos as a substrate, this investigation has compared the infection of continuous monolayer SPEV, Vero E6, and vaccine line Vero (B) cell cultures with TBEV strains of the Siberian and Far Eastern genetic types dominating in the endemic regions of Russia. After cell infection with Far Eastern (Sofyin and 205 strains) or Siberian (Aina, 2530, 2689, and 2703 strains) TBEV genetic types, the viable TBEV titers reached 2.8 Ig CPD50 for Vero (B) cells, 5.5 Ig CPD50 for Vero E6 cells, and up to 9 Ig CPD50 for SPEV cells. The quantitative scores of TBEV E antigen in enzyme immunoassay (EIA) and genome equivalents by reverse-transcription polymerase chain reaction (PCR), followed by real-time PCR, permitted one to estimate as high as 108 virions in 1 ml of culture fluid, which corresponded to those of the microscopic observations of CPD for SPEV cells and substantially exceeded the values for Vero E6 cells, and for Vero (B) cells in particular. The data of TBEV strain titration, EIA, and realtime reverse-transcription PCR suggest that the Russian vaccine Vero (B) cell line defined as meeting the WHO requirements, as well as Vero E6 cells may be used to design tick-borne encephalitis vaccine.

The antiviral activity of Ingavirin against human metapneumovirus (HMPV) infection was investigated in vitro. The investigation used the human cell line ChangConjunctiva, permissive for HMPV, clone 1-5C4, and the HMPV strain isolated at the D. I. Ivanovsky Research Institute of Virology. The experimental studies suggest that when added at a concentration of 50 to 500 microg/ml to a nutrient medium 24 hours after HMPV infection, Ingavirin suppressed effectively virus replication by 2.2-3.3 logs, respectively. When used at a concentration of 500 microg/ml 24 hours before cell infection, Ingavirin protected cells from HMPV infection.

Enzyme immunoassay of tick-borne encephalitis virus (TBE) in the samples of Ixodes ticks collected in the outskirts of the settlement of Manzherok, Maiminsk District, Republic of Altai, revealed TBE antigen in 16.9 +/- 1.9% of the talga ticks. Real-time reverse transcription-polymerase chain reaction (RT-PCR) with specific fluorescent probes and phylogenetic analysis of the nucleotide sequences of RT-RCR products corresponding to 5'-terminal fragment of the E gene of TBE, all the virus strains isolated from the ticks collected in Gomyi Altai were referred to as the Siberian genetic type that was dominant in virus-carrying ticks in the majority of endemic areas of Russia and near abroad. Viral load assays using the real-time RT-PCR with the probes indicated the threshold cycles Ct = 25.34-28.98, which, with regard to the efficiency of RNA identification and reverse transcription, was equal to about 10(4)-10(5) viral RNA copies per tick.

Six nuclear polyhedrosis virus (NPV) isolates have been isolated from dead larvae of gypsy moth in Western Siberia. Heterogeneity of virulence and reproduction activity was revealed for the NPV isolated by bioassay with Lymantria dispar L. larvae. The findings may suggest phenotypic variation of the NPV isolates. No correlation was found between virulence and reproductive activity with the only exception--the isolate Karassuk with a high virulence and a high reproductive activity. Nucleotide sequences of PCR products with primers specific to the polyhedrin gene were determined for NPV isolated Karassuk and Tatarskyi with the maximum and minimal virulence, respectively. Alignment of the nucleotide sequences demonstrated a high homology of the study polyhedrin gene fragment between NPV Western-Siberian isolates and NPV strains from the USA with two point mutations. The mutations were identical for the NPV isolated from Russia but were different from the known structures of the polyhedrin gene of the American strains. The only one from two found mutations resulted in amino acid substitution in polyhedrin protein. Consequently, the structure of both polyhedrin and encoded protein did not influence on the NPV virulence and reproductive activity.

Fractionation of RNA isolated from the inactivated purified concentrated vaccine "EnceVir" ("Virion", Tomsk, Russia) allowed the authors to reveal predominantly double-stranded RNA and trace quantities of single-stranded RNA sizing mainly less than 200 n. The fragments of tick-borne encephalitis virus (TBEV) were detected among the total RNA by the reverse transcription test, followed by nested polymerase chain reaction with the primers specific to the E gene of TBEV. The findings may suggest that immunity may be induced through activation of Th1 and Th2 after immunization with such an inactivated vaccine for effective elimination of intracellular infection and intracellular virions, respectively.

The detection rate of DNA of nuclear polyedrosis virus in the caterpillar organism ranged from 28.6 to 73.2%, the detection rate of virion antigens ranged from 10.3 to 38.2%, and that of the virus polyhedron antigens ranged from 16.1 to 52.3% in 4 natural populations of gipsy moths in the territory of the Novosibirsk region at the outbreak culmination time of a mass reproduction of the insects. An artificial activation of virus reproduction, in experiment, caused death of the insects at a level correlating with the detection rate of virion antigens and of polyhedron in a majority of the populations. The spontaneous viral infection was observed by far less seldom than the detection rate of viral DNA, virion antigen and polyhedron among the insects of 3 populations, in which damages to the forage reserve were single and did not exceed 50%. As for the insects of the 4th population, where the damage to the forage reserves amounted to 70% during 2 consecutive years, the spontaneous viral infection was higher than the detection rate of viral DNA, virion antigens and virus polyhedron. The authors believe that the trophic factor in the 4th population had a decisive impact on the activation of viral infection.

Antigenic structure of tick-borne encephalitis virus proteins was studied by ELISA with monoclonal antibodies (MAb) to E and NS1 glycoproteins of strain Sofyin. Envelope proteins appeared to be conservative which corresponded to previously determined nucleotide sequences of E gene fragments and deduced primary structures of the corresponding E protein. Five of six studied MAb to NS1 nonstructural glycoprotein of strain Sofyin reacted with this protein of all studied strains. The only exception was MAb 17C3 which discriminates West Siberian strains from Far Eastern strain Sofyin.

Binding of tick-borne encephalitis (TBE) virus RNA to proteins was studied by 3 methods: gel retardation, RNA-protein cross-linking under UV light, and RNA-protein blotting. Two cellular proteins with molecular weights about 30 and 22 kDa were detected in the nuclear fraction of two cell strains (PS and RH) and in blood mononuclear cells of patients with TBE. Weak interaction between the studied RNA and viral proteins can result from trace amounts of TBE virus proteins in infected cells and by lack of additional binding factors. Bacterial super-products and affinity chromatography for isolation of native glycoproteins were used to increase the amount of viral proteins. Purified E and NS1 proteins did not react with viral RNA, while incubation with recombinant nonstructural TBE virus NS3 protein led to a shift in the mobility of TBE virus RNA in gel in the absence of cross-linking under conditions of UV exposure after addition of heparin. Poor binding of nonstructural TBE virus NS3 protein is based on electrostatic binding. Oligoribonucleotide 14 nucleotide residues long corresponding to 5'-terminal of TBE virus genome did not react with NS3 protein, probably because of its small size or absence of certain sequences.

Tick-borne encephalitis (TBE) virus strains were isolated in West Siberia in the forest-steppe region near the Ob river in 1981-1992. Hybridization of genome RNA of 46 TBE strains with [32P]cDNA of TBE Sofyin strain revealed essential differences in the genomes of West-Siberian and Far-Eastern Sofyin strains of TBE virus. Nucleotide sequences of 6 TBE strains (1348-1503 n.) have been determined. A 89-98% homology of Siberian TBE strains has been shown, while the similarity of the respective fragment of E gene for West Siberian and Sofyin strains was no more than 81%. No significant changes in E gene of TBE strains have been detected over a 12-year period.

Affinity chromatography of lysates of continuous porcine embryo cells infected with tick-borne encephalitis (TBE) virus on sepharose with immobilized monoclonal antibodies to TBE virus proteins NS5 and NS3 results in isolation of a stable protein complex. This complex contains viral proteins NS5, NS3, p49, and, probably, two more cell proteins. This complex is not detected at the early stage of infection, and 24 h after infection its structure does not depend on the specificity of immobilized monoclonal antibodies used in affinity chromatography and on the time elapsed after the infection. Immunoprecipitates of infected cells phosphorylated TBE virus protein NS5 in vitro, but possessed no RNA-polymerase activity. Elution of the complex with buffers with pH 3.5 or 11.1 or with 2M urea failed to purify the active replicase. The complex of viral and cellular proteins isolated by affinity chromatography on different immunoadsorbents did not possess RNA-polymerase activity because of inactivation or absence of additional subunit(s).

The heterocomplex of glycoproteins E and NS1 of tick-borne encephalitis (TBE) virus was isolated from culture fluid of continuous SPEV (porcine embryo kidney) cells by affinity chromatography on CL4B sepharose with immobilized monoclonal antibodies to TBE virus protein NS1. Comparison of the TBE E-NS1 heterocomplex from culture fluid and cells showed the predominance of secreted extracellular form. A similar TBE virus heterocomplex E-NS1 was revealed in the blood sera of patients with TBE but not in their lymphocytes. Extracellular localization of the E-NS1 complex at the late stage of infection rules out its hypothesized participation in the replication of TBE virus. Virtual absence of the heterocomplex and presence of proteins E and NS1 in high concentrations in the cells makes the nonspecific aggregation of these proteins impossible. Evidently, the extracellular heterocomplex E-NS1 may react with virusspecific antibodies or with cytotoxic T lymphocytes.