Phylodynamic characteristics of reassortant DS-1-like G3P[8]-strains of rotavirus type A isolated in Nizhny Novgorod (Russia).

Since 2013, there has been an increase in reports of the spread of a double intergroup reassortant strain of rotavirus type A (RVA) with the genotype G3P[8] and other genes belonging to the second genogroup I2-R2-C2-M2-A2-N2-T2-E2-H2. In our study, we provide a molecular genetic characterization of rotaviruses with genotype G3P[8]-I2 isolated in Nizhny Novgorod. In our study, we used RT-PCR, Sanger sequencing, RNA-PAGE methods. Phylogenetic and phylodynamic analysis were performed using the Bayesian approach. According to our study, there was a significant increase in the proportion of G3P[8] from 15% during the period of 2020-2021 to 53% during the period of 2021-2022 in Nizhny Novgorod, Russia. Phylogenetic analysis based on the VP4 gene revealed that DS-1-like RVAs isolated in Nizhny Novgorod belong to different clusters of the P[8]-3.1 lineage, with a level of variation ranging from 1.1% to 1.3%. Based on the VP6 gene, the equine-like RVAs identified by us carry genetic variants belonging to three distinct clusters of the lineage I2-V, with a variation level ranging from 2.0% to 4.5%. These data indicate the genotypic diversity of circulating DS-1-like G3 RVAs. Phylogenetic analysis of the VP7 gene allowed us to assign the isolates identified in our study to the G3-1 lineage. We estimated that the circulation of the most recent common ancestor of the spreading strains dates back to 2002. Additionally, we determined the typical level of mutations in the VP7 gene, which amounted to 2.14*10-3 substitutions/per site/per year.

[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)].

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.

An increase in prevalence of recombinant GII.3[P12] norovirus in sporadic acute diarrhea in children in Nizhny Novgorod, Russia, 2018-2021.

Noroviruses are important etiological agents causing acute intestinal infection in humans. In the last decades, the most common norovirus genotype was GII.4 despite a significant genetic diversity among strains, while the active circulation of noroviruses with other genotypes was observed periodically. This study shows an increase in the detection rate of recombinant GII.3[P12] norovirus in Nizhny Novgorod, Russia, from 6.8% in 2018-2019 to 34.9% in 2020-2021. We performed a phylogenetic analysis based on the nucleotide sequences of noroviruses possessing this genotype obtained in this work, as well as presented in the GenBank database. It has been shown that the circulation of GII.3[P12] noroviruses in the study area was the result of several independent introductions, either directly from the Western Pacific region, or through the Asian part of Russia. The polyphyletic origin, the geographical expansion, and the growth of the epidemic significance of the recombinant GII.3[P12] noroviruses were noted.

[Detection SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus: Sarbecovirus) in children with acute intestinal infection in Nizhny Novgorod during 2020-2021].

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.

[Molecular monitoring of the rotavirus (Reoviridae: Sedoreovirinae: Rotavirus: Rotavirus A) strains circulating in Nizhny Novgorod (2012-2020): detection of the strains with the new genetic features].

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.

Long-term monitoring of G1P[8] rotaviruses circulating without vaccine pressure in Nizhny Novgorod, Russia, 1984-2019.

The G1P[8] genotype is one of the most common among rotaviruses circulating in the last 40 years. Therefore, this genotype is a component of rotavirus vaccines licensed throughout the world. This paper presents the results of a 35-year (1984-2019) observation of the circulation of G1P[8] rotaviruses among children under 14 in one region (Nizhny Novgorod, Russia) without vaccine pressure. Several complementary approaches were used: RNA electropherotyping by polyacrylamide gel electrophoresis, PCR genotyping, and cDNA sequencing of rotavirus VP4 and VP7 genes. A total of 8375 rotavirus-positive samples were examined, and the proportion of genotype G1P[8] rotaviruses was 39.9% (4.3-98.9%). Two cycles of high circulation activity (1984-1993 and 1993-2007) and one cycle of low activity (2007-2019) were noted. Phylogenetic analysis revealed the presence of rotaviruses of two VP4 gene lineages (P[8]-1 and P[8]-3) and two VP7 gene lineages (sublineages IA, IB, ID, II-B, II-C, and II- E). The prolonged circulation of rotaviruses of only one sublineage (G1-II-E) and then a change of the prevailing sublineage within the G1-II lineage (from E to C) during the active circulation were shown. Since 2011, when the circulation intensity of G1P[8] rotaviruses was low, the appearance of strains of the G1-I lineage and their co-circulation with strains of the G1-II lineage were observed in the population.

[Identification of rotavirus I- and E-genotypes by multiplex PCR method.]

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.

Phylogenetic comparison of the VP7, VP4, VP6, and NSP4 genes of rotaviruses isolated from children in Nizhny Novgorod, Russia, 2015-2016, with cogent genes of the Rotarix and RotaTeq vaccine strains.

Group A rotaviruses (RVA) are one of the leading causes of gastroenteritis in young children worldwide. The introduction of universal mass vaccination around the world has contributed to a reduction in hospitalizations and outpatient visits associated with rotavirus infection. Continued surveillance of RVA strains is needed to determine long-term effects of vaccine introduction. In the present work, we carried out the analysis of the genotypic diversity of RVA strains isolated in Nizhny Novgorod (Russia) during the 2015-2016 epidemic season. Also we conducted a comparative analysis of the amino acid sequences of T-cell epitopes of wild-type and vaccine (RotaTeq and Rotarix) strains. In total, 1461 samples were examined. RVAs were detected in 30.4% of cases. Rotaviruses with genotype G9P[8] (40.5%) dominated in the 2015-16 epidemic season. Additionally, RVAs with the following genotypes were detected: G4P[8] (25.4%), G1P[8] (13%), G2P[4] (3.2%). Rotaviruses with genotypes G3P[9], G6P[9], and G1P[9] totaled 3%. The number of partially typed and untyped RVA samples was 66 (14.9%). The findings of a RVA of G6P[9] genotype in Russia were an original observation. Our analysis of VP6 and NSP4 T-cell epitopes showed highly conserved amino acid sequences. The found differences seem not to be caused by the immune pressure but were rather related to the genotypic affiliations of the proteins. Vaccination against rotavirus infection is not included in the national vaccination schedule in Russia. Monitoring of the genotypic and antigenic diversity of contemporary RVA will allow providing a comparative analysis of wild-type strains in areas with and without vaccine campaign.

Predominance of new G9P[8] rotaviruses closely related to Turkish strains in Nizhny Novgorod (Russia).

Genotype G9P[8] rotaviruses are rare in the territory of Russia. They were found in Nizhny Novgorod only in 2011-2012 for the first time, when their proportion was 25.9%. During the next two seasons, G9P[8] strains were detected in only 1.8% of cases. Their proportion substantially increased again in 2014, and they became predominant in the city by 2016. Phylogenetic analysis on the basis of gene VP7 nucleotide sequences showed that this increase was accompanied by the emergence of new strains in the population. These isolates were related to Turkish strains, but not to Russian ones detected earlier.

Detection and molecular characterization of reassortant DS-1-like G1P [8] strains of rotavirus A.

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.

Comparative characteristics of the VP7 and VP4 antigenic epitopes of the rotaviruses circulating in Russia (Nizhny Novgorod) and the Rotarix and RotaTeq vaccines.

Two live, attenuated rotavirus A (RVA) vaccines, Rotarix and RotaTeq, have been successfully introduced into national immunization programs worldwide. The parent strains of both vaccines were obtained more than 30 years ago. Nonetheless, only very limited data are available on the molecular similarity of the vaccine strains and their genetic relationships to the wild-type strains circulating within the territory of Russian Federation. In this study, we have determined the nucleotide sequences of the genes encoding the viral proteins VP7 and VP4 (the globular domain VP8*) of vaccine strains and natural isolates of rotaviruses in Nizhny Novgorod, Russia. The VP7 and VP4 proteins contain antigenic sites that are the main targets of neutralizing antibodies. Phylogenetic analysis based on VP4 and VP7 showed that the majority of the natural RVA isolates from Nizhny Novgorod and the vaccine strains belong to different clusters. Four amino acids within the VP7 antigenic sites were common in both the wild-type and vaccine strains. The largest number of amino acid differences was found between the vaccine strain Rotarix and the Nizhny Novgorod G2 strains (19 residues out of 29). From 3 to 5 amino acid differences per strain were identified in the antigenic sites of VP4 (domain VP8*) between wild-type strains and the vaccine RotaTeq, and 6-8 substitutions were found when they were compared with the vaccine strain Rotarix. For the first time, immunodominant T-cell epitopes of VP7 were analyzed, and differences in the sequences between the vaccine and the wild-type strains were found. The accumulation of amino acid substitutions in the VP7 and VP4 antigenic sites may potentially reduce the immune protection of vaccinated children from wild-type strains of rotavirus.

Rotavirus infection in children of Nizhny Novgorod, Russia: the gradual change of the virus allele from P[8]-1 to P[8]-3 in the period 1984-2010.

A collection of rotavirus samples collected over a 26-year period was examined to study the dynamics of change in RV strains of genotype P[8] in a geographically defined population (Nizhny Novgorod, Russia; children under 6 years) with no vaccine pressure. Phylogenetic analysis of gene VP4 (subunit VP8*) showed the presence of two lines of genotype P[8]: P[8]-1 and P[8]-3. Since 1997, the dominant population of rotavirus has been occupied by strains carrying the allele P[8]-3, which is associated with G1, G3 and G4. The complete replacement of the allele P[8]-1 to P[8]-3 took 19 epidemic years.