Re-emergence and Spread of Norovirus Genotype Gii.17 Variant C in 2021-2023

Re-emergence and Spread of Norovirus Genotype Gii.17 Variant C in 2021-2023 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Re-emergence and Spread of Norovirus Genotype Gii.17 Variant C in 2021-2023 Epifanova N.V., Oparina S.V., Morozova O.V., Sashina T.A., Alekseeva A.E., and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5393670/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Norovirus is one of the leading causes of acute gastroenteritis worldwide and is characterized by significant genetic diversity. In this study, based on phylogenetic analysis of the genome sequences of noroviruses circulating in Nizhny Novgorod in 2014–2023, as well as those retrieved from the GenBank database, the return to active circulation of the C variant of the GII.17[P17] genotype, displaced in 2015–2016 by the D variant, is shown. A new subvariant C2, different from the C1 subvariant circulating in the middle of the last decade, was identified. Amino acid substitutions characteristic of C2 were found in the main structural protein VP1, bringing it closer to the Tokyo_JP_1976 strain identified in the 1970s. It was established that the C2 subvariant circulated in 2021–2023 in European and American countries and caused outbreaks of norovirus infection. The data obtained indicate that the evolution of the phylogenetic lineage represented by the C variant of the GII.17 genotype has been continuing in the last decade and has the character of convergence with the ancestral strain, and for four years (2017–2020) these processes were latent. Virology Norovirus Genetic diversity Genotyping Phylogenetic analysis Variant Subvariant Figures Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Noroviruses (NoVs) discovered more than half a century ago [1] belong to the family Caliciviridae , genus Norovirus ( Viruses; Riboviria; Orthornavirae; Pisuviricota; Pisoniviricetes; Picornavirales; Caliciviridae ).Currently, NoVs is associated with one fifth of all aсute gastroenteritis cases in the world and is estimated to cause over 200,000 deaths annually in developing countries [2]. NoVs is also the main etiological agent of outbreaks of non-bacterial gastroenteritis in kindergartens, schools, hospitals, camps, cruise ships, military units and nursing homes, affecting all age groups of the population [3]. Noroviruses are small, non–enveloped viruses (size: 38 nm), the capsid of which consists of 90 dimers of the major structural capsid protein VP1 and several molecules of the minor capsid protein VP2 [4]. The human norovirus genome is a linear single-stranded (+) RNA approximately 7.5–7.7 thousand nucleotide bases (nt) in length with a polyadenylated 3'-end and a VPg peptide covalently linked to the 5- end, and is organized into three open reading frames (ORFs) [5]. ORF1 encodes a large polyprotein, the precursor of six nonstructural proteins (NS1/2 - NS7), including RNA-dependent RNA polymerase (RdRp, NS7), ORF2 overlaps at 14–20 nt with ORF1 and is usually translated from subgenomic RNA [4]. ORF2 encodes the major structural capsid protein VP1, which is subdivided into the N domain, the S domain forming the virion shell, and the P domain protruding above the shell surface [6]. The P domain consists of two relatively conservative subdomains P1-1 and P1-2, between which the highly variable P2 subdomain is located. P2 carries antigenic determinants and binding sites to host cell receptors (co-receptors) – blood group antigens represented on intestinal enterocytes (histo-blood group antigens, HBGA) [7]. HBGA are complex fucose-containing glycans that define polymorphic human blood groups and play a critical role in susceptibility to noroviruses [8, 9]. ORF3 encodes the minor structural capsid protein VP2 and overlaps with ORF2 at 1 nt [4]. VP2 is located inside the viral particle and participates in capsid assembly and genome encapsulation [10, 11]. At the junction of the reading frames encoding non-structural and structural proteins, there is a “hot spot” where recombinations often occur in the norovirus genome, so a dual nomenclature is currently adopted that takes into account norovirus genotypes in two reading frames. The ORF1 region encoding RNA polymerase and the ORF2 region encoding the N/S domain and P domain of the capsid protein VP1 are most frequently used for typing [12]. According to the classification presented in 2019, NoVs divided into ten genogroups (GI-GX). Based on the analysis of the amino acid sequence of the main capsid protein VP1, 48 genotypes are isolated, based on the analysis of the nucleotide diversity in the RdRp region, noroviruses are divided into 60 P-types [13]. Some genotypes and P-types, for which single nucleotide sequences still exist, are not assigned numbers, they are called NA. Mutations that cause changes, insertions or deletions of amino acids in VP1 change the properties of the virus, affect the communication and formation of the immune system and lead to the appearance of new antigenic variants of NoVs [14, 15]. Worldwide, genotype GII.4 is responsible for most outbreaks and sporadic cases of gastroenteritis associated with NoVs since the 1990s [16]. The circulation of noroviruses of other genotypes periodically intensifies, acquiring epidemic distribution [17, 18, 19]. GII.17 viruses have been detected in the world for more than four decades. The earliest are noroviruses identified in the study of archival samples of copromaterial collected in 1978 in French Guiana [20] and in 1976 and 1982 in Japan [21]. Until 2012, cases of GII.17 registration worldwide were rare [22]. One of the few exceptions was the identification of the GII.17 genotype in 76% of noroviruses detected in 2012–2013 in surface waters of rivers in rural and urban areas of Kenya [23]. However, in the winter of 2014–2015, the number of outbreaks caused by the GII.17 genotype increased sharply in Southern China [24, 25], Hong Kong [26], Japan [27] and South Korea [28]. An outbreak outside Asia caused by this genotype was reported in Romania in October-December 2015 [29]. In Russia, aquatic outbreaks of norovirus infection caused by GII.17 were described in the Khabarovsk Krai in March-June 2015 [30] and in the Republic of North Ossetia-Alania in June 2015 [31]. GII.17 norovirus has not only caused outbreaks but has also been sporadically detected in China (Shanghai, Taiwan) [32, 33], the United States of America [34], and Latin America [35, 36, 37, 38]. Currently, GII.17 noroviruses are divided into four variants – A, B, C and D [39]. A and B variants include noroviruses detected in isolated cases since the 70s of the last century in different countries of the world and having a polymerase gene with specificity P4, P3, P13, P16, P31 [22]. C variant, which appeared in 2012, had a polymerase type that had not previously been detected in association with other VP1 genotypes, and was designated P17 [27]. Somewhat later, D variant with the same P17 polymerase emerged and has spread widely around the world since 2014–2015, soon completely replacing C variant [40]. The new GII.17 variants (C and D) were found to have stronger HBGA binding ability than the previous variants (A and B) [9, 41, 42]. It was assumed that GII.17 norovirus may have pandemic potential and replace genotype GII.4, the epidemic variants of which have dominated the world since the mid-1990s [22]. However, later the intensity of GII.17 circulation decreased everywhere, and only in 2022–2023 we noticed an increase in its share in the spectrum of norovirus genotypes circulating in Nizhny Novgorod. The aim of the work is the molecular genetic characteristics of GII.17 noroviruses, identified in Nizhny Novgorod, in comparison with the dynamics of circulation of noroviruses of this genotype in the world. Materials and methods Samples under study. The study used fecal samples from children hospitalized with symptoms of acute intestinal infection in the children's infectious diseases hospital of Nizhny Novgorod in the period 2014–2023. RNA was extracted and precipitated using a RIBO-prep reagent kit (Central Research Institute of Epidemiology [CRIE], Russia). Norovirus was detected using a real-time PCR diagnostic kit (AmpliSens Rotavirus/Norovirus/Astrovirus-FL and AmpliSens OKI viro-screen-FL", CRIE). Virus cDNA for genotyping and amplification before sequencing was obtained using a REVERTA-L kit [CRIE]. Sequencing of the norovirus genome . To determine the genotype of noroviruses, cDNA sequencing was performed on a 570-nt region of the viral genome, including the overlap of open reading frames for nonstructural and structural proteins and encoding the following regions: the C-terminal region of RNA-dependent RNA polymerase and the N-terminal region of the S-domain of the capsid protein VP1 (RdRp-VP1). The method based on a combination of two pairs of previously published primers [43, 44], was proposed by Cannon J.L. et al. [45]. For cDNA amplification, we used reagents manufactured by Sileks Ltd., Evrogen Ltd. Syntol Ltd. (Russia) and primers synthesized in Syntol Ltd. and Evrogen Ltd. (Russia). To determine the complete sequence of the structural protein gene VP1 of GII.17 norovirus, amplification of three overlapping fragments was carried out using three pairs of specially selected primers (Table 1). Fragments for sequencing were purified using DNA fragment purification kits from gel (FractalBio Ltd., Russia). Determination of the primary structure of cDNA fragments of the norovirus genome was carried out by the Sanger method in automatic mode on genetic analyzers Beckman Coulter GenomeLab GeXP (Beckman Coulter, USA), Nanofor-05 (Institute for Analytical Instrumentation, Russia) using DTCS Quick Start Kit (Beckman Coulter, USA), BigDye Terminator v3.1 (Applied Biosystems, Thermo Fisher Scientific, USA), GenSek (Syntol, Ltd., Russia). Whole genome sequencing was performed on the Illumina platform for one norovirus isolate. To obtain norovirus whole genome cDNA, reverse transcription was performed in the presence of the Tx30SXN primer [46], specific to the 3'-end of the viral genome, using the RNAscribe RT Reverse Transcriptase reagent kit (Biolabmix Ltd., Russia). Amplification of whole-genome cDNA fragments was performed in the presence of Tx30SXN and GII_1–35 primers specific to the 3'- and 5'-ends of the viral genome [39] using the BioMaster LR HS-PCR-Color-2x kit (Biolabmix Ltd., Russia). The concentration of viral cDNA was assessed using a Qubit 2.0 fluorimeter (Invitrogen, Austria) and the Spectra Q HS kit for quantitative DNA determination (Raissol, Russia). Preparation of cDNA libraries for sequencing was performed using the ShotGun "SG GM" kit and a set of indexed primers for dual barcoding for Illumina sequencers (Raissol, Russia). Sequencing was performed on an iSeq100 instrument (Illumina, USA). The obtained archives of viral genome sequence readings were processed using the Unipro UGENE software [47], version 42.0. The GII.17 NoV whole genome from the GenBank database [48, 49] were used as a reference genome. As a result of combining short reads, a consensus sequence of the norovirus genome with coverage from 10x to 300x was obtained. Norovirus genotyping. The resulting sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) [50, 51], as well as using web services for automatic norovirus genotyping Norovirus Genotyping Tool Version 2.0 [52, 53], Calicivirus typing tool [54, 55], NoroNetRus [56, 57]. GenBank numbers . The partial nucleotide sequences obtained in this study were registered in the international GenBank database with accession numbers MK033836-MK033839, MN542836-MN542838, ON854092-ON854113, ON854111, ON854112, OR475165-OR475194, the full genome sequence – with the accession number OP712199. Phylogenetic analysis. A preliminary phylogenetic analysis of the nucleotide sequences we established was performed in the MEGA 11 program using the Maximum Likelihood method, using the Tamura-Nei substitution model [58] with five reference strains of noroviruses of the GII.17 genotype: KC597139, MW305610, AB983218, LC037415, AB684681. We then compiled a set of related sequences of norovirus genotype GII.17 from the GenBank database. The search for related sequences was performed using the BLAST. Nucleotide sequence alignment was performed using MEGA software, version 11 [58]. A 525 nt region of the RdR-VP1 viral genome was used for the analysis. A total of 485 norovirus sequences identified from 1976 to 2023 in 34 countries were downloaded from the Genbank database as of March 2024. Sequences with high homology, identical time and place of isolation were removed from the alignment. As a result, 377 sequences were included in the file for analysis, including 56 obtained in this study. Phylogenetic analysis based on sequences of complete genes VP1 (238 sequences including 15 obtained in this study), VP2 (216 sequences, including one of the Nizhny Novgorod isolate) and RdRp (215 sequences including one of the Nizhny Novgorod isolate) was carried out separately. Phylogenetic analysis and effective population size (EPS) calculation were performed using the Bayesian Skyline model [59] with the BEAUti 1.10.4 and BEAST v 1.10.4 [60] software packages. Rates of nucleotide substitution per sites and time to the most recent common ancestor (tMRCA) were estimated using Bayesian Markov chain Monte Carlo (MCMC). MCMC length was 500 million to 1 billion generations depending on the region (gene) analyzed to ensure effective sample size (ESS) values > 200. The nucleotide substitution process was estimated using the Hasegawa–Kishino–Yano (HKY) model. Runs were performed under relaxed (uncorrelated lognormal) clock model. Visualization of population dynamics and analysis of output MCMC files were estimated using Tracer v1.7.1.1. [61]. The Maximum Clade Credibility (MCC) tree was summarized from the posterior distribution of trees using TreeAnnotator v1.10.4 included in the BEAST package and visualized and annotated using FigTree v1.4.4 [62]. The amino acid variations within and between clades observed in the tree for VP1 were examined by applying the Poisson correction with MEGA 11. Modeling the structure of the VP1 P-domain and RdRp. Molecular graphics were performed using UCSF ChimeraX [63, 64, 65]. Spatial models of the VP1 protein P domain of GII.17[P17] NoV are constructed based on the P domain of the Kawasaki323_JP_2014 strain, (5F4M, accession code in the Protein Databank – PDB DOI: https://doi.org/10.2210/pdb5F4M/pdb , https://www.rcsb.org/structure/5F4M ) and on the P domain of the Kawasaki308_JP_2015 strain, (5F4O, PDB DOI: https://doi.org/10.2210/pdb5F4O/pdb , https://www.rcsb.org/structure/5F4O ). The spatial model of the RdRp GII.P17 NoV is constructed based on the RdRp GII.P4 NoV, (4QPX, PDB DOI: https://doi.org/10.2210/pdb4qpx/pdb , https://www.rcsb.org/structure/4QPX ). Results From July 2014 to June 2023, fecal samples from 14,180 children hospitalized in a pediatric infectious diseases hospital were tested for noroviruses. Noroviruses were detected in 2,438 children (17.19% of cases). The genotype was determined for 691 isolates, genotype GII.17 was identified in 70 cases (10.13%). The share contribution of genotype GII.17 to the population structure of noroviruses in the period 2014–2023 in Nizhny Novgorod . NoV GII.17 was first detected in Nizhny Novgorod at the end of the 2014–2015 season (in June 2015) and in the 2015–2016 season, it took second place in the spectrum of norovirus genotypes after GII.4, accounting for 28.1% of the number of typed isolates. Subsequently, its share fluctuated from 3.0 to 10.6% in different epidemic seasons (Fig. 1 ). The sharp decline in the detection of GII.17 norovirus in the second half of 2016 and its subsequent detection in no more than 10–11% of cases suggested that GII.17 norovirus had largely exhausted its epidemic potential and would continue to circulate at low frequency as a minor genotype. However, in the 2022–2023 season, an increase in the share of GII.17[P17] in the spectrum of detected genotypes to 36.1% was observed. Moreover, this was not accompanied by an increase in the frequency of detection of noroviruses; on the contrary, the indicator even decreased slightly compared to the previous season (13.8% − 9.9%, respectively) (Fig. 1 ). To identify the characteristics of new strains of this genotype, a philodynamic analysis was conducted based on the nucleotide sequences of the GII.17 norovirus genome circulating in Nizhny Novgorod, as well as those extracted from the GenBank database. Analysis of RdRp-VP1 fragment For four isolates of GII.17 norovirus, identified in Nizhny Novgorod in 2015–2016, partial nucleotide sequences of the N/S domain of the VP1 gene were obtained, and for 56 isolates identified in 2017–2023, sequences of the region including the overlap zone of open reading frames encoding RNA-dependent RNA polymerase and the VP1 capsid protein (RdRp-VP1 fragment ) were obtained. The latter were used to construct a phylogenetic tree with reference sequences of A (KC597139_A_C142_GF_1978), B (MW305610_B_4522_AR_2005), C (AB983218_C_Kawasaki323_JP_2014) and D (LC037415_D_Kawasaki308_JP_2015) variants, as well as the earliest isolate of genotype GII.17, identified in an archival sample of 1976 from Tokyo (Japan), carrying P3 polymerase and not assigned to any variant (AB684681_ 27 − 3/Tokyo_JP_1976). Phylogenetic analysis of the studied region (size: 525 nt) showed that the Nizhny Novgorod NoVs identified in 2017–2022 belonged to D variant together with the reference strain Kawasaki308_JP_2015. The Nizhny Novgorod isolates of 2021–2023 formed a reliable cluster with the strain Kawasaki323_JP_2014 – the prototype of C variant, although they formed a separate branch within this cluster, which we designated " C2 subvariant " in contrast to the original strain, which in this context we will call the "C1 subvariant" (Fig. 2 a). Isolate 27 − 3/Tokyo_JP_1976 turned out to be closer in this region to representatives of C and D variants than to A and B variants. Comparison of aligned fragments showed that C2 subvariant carries nucleotide substitutions that distinguish it from both the prototype C variant and the prototype D variant (Fig. 2 b). These substitutions allow identifying a new subvariant even when sequencing shorter genome regions. Thus, four Nizhny Novgorod isolates of 2015–2016, for which we obtained only regions of the N/S domain of the VP1 gene, according to this analysis belonged to D variant, since they had characteristic substitutions C379T, C473T, C479T. To clarify the origin of the new GII.17 subvariant, the sample was significantly expanded and analyzed using the Bayesian phylodynamic approach. The phylogenetic tree constructed on the basis of 377 sequences, the common ancestor for all analyzed sequences existed in 1848 (Supplementary material 1, Table 2). Variant A includes 12 sequences of noroviruses circulated in 2002–2019 in the USA, Great Britain, Guatemala, Argentina, China, Paraguay, Japan, and South Africa and carried polymerase genes with P16 and P31 specificity. The prototype strain KC597139_C142_GF, identified in 1978 in French Guiana, also belongs to this variant. It should be noted that the polymerase genotype of this strain was previously annotated as P4 [22], but in automated norovirus genotyping systems it is currently defined as P12. Variant B includes 14 sequences of noroviruses with P13 polymerase circulated in 2004–2009 in France, Cameroon, Argentina, Peru, China, Russia (Chelyabinsk), as well as 7 sequences of noroviruses detected in 2019–2020 in wastewater in Pretoria (South Africa), carrying P7 and P17 polymerase [66], and one sequence of noroviruses with P17 from Ecuador (2019). It is worth noting that isolate DQ438972_B_Katrina_US_2005, often used as a reference strain for B variant, is not included in this analysis because its polymerase gene sequence has not been determined. An intermediate position between B and C variants is occupied by sequences of three isolates from Japan (two from 1976 with P3 and P13 [21], one from 1982 [20], whose polymerase is closest to P40), one isolate from Tunisia (1977) with polymerase P13, one isolate of norovirus with polymerase P3, identified in Saudi Arabia in 1990, and one isolate from Argentina (2015) with polymerase PNA6 [67]. The divergence of C and D variants dates back to 2005 (Table 2). Variant C (91 sequences) in this tree is represented by a single branch with tMRCA, which existed in 2010. At the base of this branch is subvariant C1, from which subvariant C2 evolved (Fig. 3 , Fig. 1 supplementary materials). C1 subvariant includes noroviruses with P17 polymerase which circulated in 2012–2015 in China, Brazil, the Netherlands, Australia, India, South Korea, France, Canada, Japan (including the earliest isolate LC433694_OC12019_JP_2012, detected in Osaka in March 2012 [68] and the reference strain AB983218_C_Kawasaki323_JP_2014), as well as the only 2016 strain from the UK [69]. C2 subvariant with tMRCA that existed in 2020 includes sequences of 33 Nizhny Novgorod isolates (including 1 from 2021, 22 from 2022, and 11 from 2023), one isolate from the USA (2022), 3 from Romania (2021) [70], 13 from Brazil (2023) [71], and 3 isolates from Russia (two from Khabarovsk, 2022, and one from Kamensk-Uralsky, Sverdlovsk Region, 2023). It is noteworthy that we did not find C variant strains circulating in the period 2017–2020 in the GenBank database. Alignment of partial sequences available in GenBank encoding the N/S domain of the VP1 protein of noroviruses circulating in other regions of Russia, which were not included in this analysis, and analysis of characteristic nucleotide substitutions showed that 18 isolates obtained in the Sverdlovsk region in 2022–2023 (OP862435-OP862440, OR399139, OR399140, OR447705-OR447707, OR717543-OR717547, OR726224) also belong to the C2 subvariant (data not shown). Variant D (reference strain LC037415_D_Kawasaki308_JP_2015) includes the majority of sequences (254) in the phylogenetic tree we constructed, including sequences of twenty-three Nizhny Novgorod norovirus isolates of 2017–2022 and two isolates identified in Amursk, Khabarovsk Krai, in 2022 (OR775312, OR775313) (Fig. 1 , supplementary materials). Noroviruses of variant D, whose tMRCA according to the topology of this tree existed in 2010, circulated widely in the world in 2014–2022. The vast majority of them had polymerase P17. The exceptions were two isolates with polymerase P25 (France 2014, Netherlands 2016), 8 isolates from South Africa 2018–2020, including 6 with polymerase P7, one with P16 and one with P33 (Supplementary material 1). The overall mutation accumulation rate for the GII.17 genotype based on the analyzed genomic region was 1.78E-3 (1.09E-3 ‒ 2.55E-3) substitutions per site per year (Table 2). Individual clusters evolved at a comparable rate, with this figure being slightly higher for variant D (5.38E-3). ORF2 analysis For 15 Nizhny Novgorod isolates, complete nucleotide sequences of ORF2 encoding the major structural protein VP1 were determined. These sequences as well as the ORF2 sequences of the strain for which the whole genome sequence was obtained (OP712199_755_NN_RU_2022), were included in the sample for phylogenetic analysis of 238 sequences. The topology of the obtained phylogenetic tree is generally similar to that for the RdR-VP1 fragment (see Supplementary materials 2). Clusters of A (12 sequences), B (11 sequences), D (174 sequences, including three Nizhny Novgorod isolates from 2020) variants are clearly distinguished. Strains AB684681_P3_27 − 3/Tokyo_JP_1976 and MW305625_PNA6_ Arg13099_AR_2015 also occupy an intermediate position between B and C variants. The common ancestor for all analyzed VP1 gene sequences existed in 1806 (Table 3). However, unlike the tree for the RdRp-VP1 fragment, in this phylogenetic tree C variant (41 sequences) is heterogeneous (Fig. 4 ). The strain MH218689_231_GB_2016, as well as the strains MK907790_G19_020_FR_2014 and MW661253_BMH15-067_Ca_2015 separated from C1 subvariant, which had tRMCA in 2009. The latter in this tree occupy an intermediate position between C1 and C2 variants and are designated "C1/2 subvariant", their common ancestor with isolates of C2 subvariant existed in 2011. C2 subvariant is formed by 13 Nizhny Novgorod isolates from 2021–2023, 3 isolates from Romania (2021) and one from the USA (2022), whose tMRCA dates back to 2017. The evolution rate of D variant and C2 subvariant based on the VP1 gene (5.78E-3 and 4.7E-3, respectively) significantly exceeded this indicator for the genotype as a whole (1.90E-3) (Table 3). Based on the obtained alignment of nucleotide sequences, the derived amino acid sequences were obtained by translation in the MEGA program. For the VP1 protein, the differences between the amino acid sequences of representatives of different variants and subvariants were determined using the Poisson correction. Figure 5 shows a histogram of the distribution of differences, expressed as a percentage, relative to the reference strain of C variant, C1 subvariant, AB983218_C_Kawasaki323_JP_2014. It can be seen that within C1 subvariant the indicator ranges from 0 to 0.19% (on average 0.16%), for isolates of C2 subvariant the range varies from 2.07 to 2.64% (on average 2.26%). For strains MK907790_G19_020_FR_2014 and MW661253_BMH15-067_Ca_2015, which, in our opinion, are an intermediate link between C1 and C2 subvariants (C1/2 subvariant), the differences from the prototype strain are 1.68% and 1.87%, respectively. The differences with representatives of D variant range from 4.18 to 4.76 (on average 4.42%), A variant – from 10.20 to 11.45 (on average 10.69%), B variant – from 12.48 to 12.90 (on average 12.76%) Taking into account the fact that the criterion for defining a new variant adopted for the GII.4 genotype (maximum amino acid variability within a variant is 4.1% [40]) was adapted for GII.17, isolates that differ from the prototype strain C by only 2.26% should be classified as belonging to the same variant. When aligning the amino acid sequences of representatives of all four variants, it was found that the new subvariant, compared to A and B variants, has deletions at positions 295, 296, and 385 and an insertion/substitution at 346/346A, as do representatives of C and D variants (data not shown). It should be noted that in publications by different authors there are some shifts in the position of deletions and insertions depending on the alignment performed. These positions are indicated in accordance with the alignment presented in the article by Sang et al. (2018) [39]. Alignment of the new C2 subvariant sequences with only the VP1 amino acid sequences of C and D variants revealed differences with C1 variant at eleven amino acids, with no insertions or deletions (Fig. 6 ). One substitution is located in the S domain, three in the P1 subdomain, and seven in the P2 subdomain. There are 30 differences between the representatives of the new subvariant and the D variant, including two deletions at positions 373 and 397, and 28 amino acid substitutions, of which two are located in the S domain, four in the P1 subdomain, and 25 in the P2 subdomain (Fig. 6 ). The following substitutions are characteristic of C2: 296N, 297P, 361R, 372K, 385/384R, 411/409V, 433/431L, 437/435S (the positions are indicated for D variant and subvariant C2, respectively, taking into account deletions). The substitutions 296N, 297P and 385/384R are also present in the strain AB684681_P3_Tokyo_JP_1976, which, according to the phylogenetic analysis, is close to C and D variants, the substitution 411/409V was found in one strain of C1 subvariant - OP205532_R02-06_NL_2015 (Fig. 6 ). The 433L substitution is present in two strains D variant from USA (MT344181_118-3_US_2015 and MT371769_PNA_147-2_US_2015) (data not shown). At position 377/376, both the amino acid N, found in most strains of the C1 subvariant, and the D, characteristic of the D variant, are present in different representatives of the C2 subvariant. Only the 361R, 372K, and 437/435S substitutions are unique to C2 (Fig. 6 ). The strains of subvariant C1/2 occupy an intermediate position, having amino acids similar to variant C1 in three cases (334V, 433/431F, 437/435F), in six cases – to the new subvariant C2 (144I, 296N, 297P, 385/384R, 411/409V, 449/447I), in one case – as in individual representatives of C1, C2 and all representatives of variant D (377/376D). The strain. The only unique substitution for C1/2 subvariant is 361K (Fig. 6 ). Figure 6 shows the positions of amino acids that make up the conformational epitopes identified in GII.4 noroviruses – A, C, D, E, the location of which is mapped by analogy with those identified for GII.4 ([14, 72, 73], and also taking into account some experimental data obtained for GII.17 [74, 75, 76, 77]. It is evident that the amino acid substitutions characteristic of C2 subvariant are found in epitopes A (296, 297) and E (411/409) and may cause differences in antigenicity from both C1 subvariant and D variant. Five amino acid residues that typically interact with α-1,2-fucose of HBGA (348/348T, 349/349R, 378/377D, 443/441G, 444/442Y for D and C variants, respectively, indicated by asterisks and pink shading in Fig. 6 ) were highly conserved and identical for all representatives of variants C and D, and were also present in strain AB684681_P3_Tokyo_JP_1976. The amino acid substitutions we identified for VP1 of C2 NoV subvariant were plotted on the following structural models: P-domain of GII.17 norovirus Kawasaki323 (C1 variant), and P-domain of GII.17 norovirus Kawasaki308 (D variant) [74]. The resulting spatial models clearly demonstrate a relatively small number of amino acid substitutions compared to subvariant C1 and multiple substitutions compared to D variant (Fig. 7 ). ORF3 analysis For one Nizhny Novgorod isolate, a whole genome nucleotide sequence was obtained, which allowed it to be included in the analysis of ORF3, encoding the minor capsid protein VP2. A total of 216 sequences were included in the analysis. The topology of the obtained tree is also generally similar to the trees for the VP1- RdRp region and for the VP1 gene (see Supplementary material 3). The common ancestor for all analyzed VP2 gene sequences existed in 1833 (Table 3). Differences are again observed for C variant, which is represented by two branches (Fig. 8 a). Strain MH218689_231_GB_2016 did not form a separate lineage, but entered the branch of C1 subvariant with the closest common ancestor in 2010. The second branch again split into two subbranches, one of which contains isolates from France in 2014 and from Canada in 2015 (MK907790_G19_020_FR_2014 and MW661253_BMH15-067_Ca_2015, C1/2 subvariant), and the other - C2 subvariant, which includes, as well as the VP1 tree, one Nizhny Novgorod isolate of 2022, three isolates from Romania (2021) and one from the USA (2022). The common ancestor of this branch existed in 2012, and C2 subvariant – in 2018 (Table 3). The evolution rate for the genotype as a whole based on the VP2 gene (2.72E-3) was slightly higher than based on other regions of the genome, and for D variant, C2 subvariant, and also for C and D clusters as a whole (5.97E-3, 4.65E-3, and 7.01E-3, respectively) it significantly exceeded this indicator (Table 3). According to the amino acid sequence of the VP2 protein, representatives of C2 subvariant have 9 substitutions compared to C1 variant, and 12 compared to D variant (Fig. 8 ). At positions 145 and 161 of the new subvariant, the presence of amino acids characteristic of some isolates of variant B (S) is observed, and at position 243 – for variants A and B (R), at position 146 – for the reference strain of variant A (S) (data not shown). In a number of positions, representatives of the new subvariant have unique substitutions – 30S, 58N, 145G. Substitutions 140S and 186R are present only in strains from Romania (Fig. 8 b). ORF1 analysis The most important nonstructural protein encoded by ORF1 is RNA-dependent RNA polymerase (RdRp). The complete polymerase gene was analyzed for 215 sequences (Supplementary materials 4) (including one from Nizhny Novgorod). The sequences are divided by the type specificity of the polymerase and grouped by variants: P12, P16, P31 –A variant, P13 – B variant, P17 – C and D variants. Sequences with the type specificity P3, P25, PNA6 occupy an intermediate position. The common ancestor for the polymerase genes existed 100 years earlier (in 1733) than for the structural protein genes, which is explained by the different type affiliation of the analyzed genes, compared to the single type affiliation of the VP1 and VP2 genes (GII.17) (Table 3). Variant C for the polymerase gene is represented by two branches - the first is subvariant C1, the second branch again split into two subbranches, which are formed by the same isolates as in the trees for ORF2 and ORF3 - subvariant C1/2 (isolates from France in 2014 and from Canada in 2015) and subvariant C2 (1 isolate from Nizhny Novgorod in 2022, 3 isolates from Romania (2021) and one from the USA (2022)) (Fig. 9 a). Moreover, the time of existence of the closest common ancestors for the clusters presented in the table (except for the genotype as a whole) practically coincides with the genes of structural proteins (Table 3). The evolution rate of D variant and C2 subvariant based on the RdRp gene (3.99E-3 and 3.17 E3, respectively) slightly exceeded this indicator for the genotype as a whole (1.70 E-3) (Table 3). Analysis of amino acid substitutions in the P17 polymerase sequence in comparison with Kawasaki323 showed that most representatives of C variant (including the new C2 subvariant) have amino acid L at position 206, unlike the prototype strain, which carries F at this position. F at position 206 is also present in all representatives of D variant (Fig. 9 b). At position 88, strain Kawasaki 323 has R, just like of subvariant C1/2 strains, unlike all other representatives of C and D variants. The five analyzed isolates of the new subvariant, compared to the representatives of C1 and D variants, are characterized by the substitutions R49K, N79K, N81S, Q102E, I215V (the last one – with the exception of the strain from the USA) and A293V (in the strain from the USA – A293T). Similar substitutions at positions 79 and 102 are present in two isolates of C1/2 subvariant, while the substitution V395I was found only in the sequences of the Romanian isolates. The amino acid substitutions of Rdp GII.17 norovirus C2 subvariant were mapped onto the structural model of GII.P4 norovirus polymerase [78] (Fig. 9 c). It is evident that the substitutions at positions 206 and 215 are located at the hydrophobic cleft near the active center of the polymerase. When aligning the deduced amino acid sequences encoding other nonstructural proteins, substitutions characteristic of the C2 subvariant were noted in the region of protein P48 (56T, 58R, 85A, 195N, 198A, 276V, 281K, 286M), NTPase (453S, 464V), P22 (754T, 794 T/A, 830T) and VPg (966R) (Fig. 10 ). In the protease region, only the I1056M substitution is unique, present in two isolates of the C1/2 subvariant. Demographic analysis Skyplot Demographic inference using the Bayesian skyline plot (BSP) was performed on samples for the RdRp-VP1 region and the complete VP1, VP2, RdRp genes (Fig. 11 ). All four plots show a steady decline in the effective population size (EPS), which became apparent after 2005. Around 2015, a peak in EPS growth was noted, which for structural and non-structural proteins was shifted by 1 year, and for the RdRp-VP1 region it was extended by two years, after which this value decreased again and a tendency to reach a plateau was observed. The emergence of new C 1/2 and C2 subvariants did not affect the EPS value. Indeed, we did not observe an increase in the frequency of norovirus detection in the 2022–2023 season; on the contrary, there was a slight decrease in this indicator compared to the previous season (from 13.8–9.9%, respectively). Discussion GII.17 noroviruses, which caused an increase in the incidence in East Asia in 2014–2015 and subsequently spread throughout the world, have been detected in a relatively small percentage of cases in recent years [79, 80, 81]. However, during the 2022–2023 season, when monitoring the circulation of noroviruses in Nizhny Novgorod, a significant increase in the share of this genotype in the genetic spectrum of the norovirus population was observed. Phylogenetic analysis of the region of the norovirus genome, including the zone of overlapping reading frames encoding non-structural and structural proteins (RdRp-VP1), showed that 377 analyzed sequences were divided into four clusters corresponding to A, B, C and D variants. At the same time, the Nizhny Novgorod isolates of 2018–2022 belong to D variant, which has been widespread in the world in recent years. C variant, in our opinion, is divided into two subvariants. We identified C1 subvariant, which, as phylogenetic analysis showed, was detected not only in Asian countries, but was present in Europe and Latin America in 2013–2016 [69, 82, 83] and was not detected in the world after 2016. Another subvariant, which included the Nizhny Novgorod isolates of 2023, as well as some isolates of 2022 and one of 2021, cluster with noroviruses from Romania (2021), the USA (2022) and Brazil (2023). Based on the topology of the phylogenetic tree constructed using the VP1-RdRp fragment, this group can be considered as a "derivative" of noroviruses of the C1 variant circulating in 2012–2016, and represent a new GII.17 C2 subvariant. Phylogenetic analysis of the VP1, VP2, and RdRp complete genes conducted on the basis of 238, 216, and 215 sequences, respectively, showed a slightly different picture: the topology of phylogenetic trees indicated that representatives of the C2 subvariant did not originate from C1, but evolved from an ancestor common to C and D variants that existed in 2003–2005. The intermediate stage in the course of this evolution were the isolates MK907790_G19_020_FR_2014 and MW661253_BMH15-067_Ca_2015, which we combined into the C1/2 subvariant. Five isolates assigned to the C2 subvariant and two – to the C1/2 subvariant were previously included in the analysis of almost whole nucleotide sequences conducted by Dinu et al. (2023) during the study of a large outbreak of norovirus infection in Romania in 2021 [70]. The authors showed that these genomes fall into a distinct P.17-polymerase-type clade/cluster, different from the previously described Kawasaki clades/clusters, but noted that it is hard to determine if the potential new clades identified in its study represent transitional variants or successful, well-established variants. The fact that these seven isolates belong to C variant, and not to distinct new variants, as suggested by Dinu (2023), is, in our opinion, confirmed by the assessment of the percentage of differences in the amino acid sequences of the VP1 protein of these isolates, which is 2.26% with representatives of C variant and significantly more with representatives of D variants (4.42%), A (10.69%) and B (12.48%). A criterion for identifying individual variants within a genotype, accounting for more than 4.1-5% differences in the complete amino acid sequence of the VP1 protein, was proposed by the International Norovirus Classification Group for GII.4 norovirus variants and extrapolated to other genotypes [16, 40, 84]. Our data showed that C2 subvariant has spread in Russia and is also circulating in Europe, the USA and Latin America (Brazil). Noroviruses, the sequences of which cluster with the Nizhny Novgorod strain ON854112_173_NN_RU_2022, judging by the phylogenetic trees presented in the article by Eftekhari M. et al. (2023), were also detected in Iran in 2021 [85]. The new subvariant has a number of substitutions in the amino acid sequence of the VP1 protein, located near conformational antigenic epitopes blocked by neutralizing antibodies. These substitutions distinguish them from both representatives of the C1 subvariant and from representatives of the D variant, which has been widespread in recent years, and may facilitate evasion of previously formed immunity to GII.17 norovirus. The critical importance of five amino acid residues (namely 293Q, 294I, 295N, 296Q and 299R) that form the blocking epitope of cluster IIIb (D variant) was highlighted by Yi et al. (2021) [76]. Liao et al. (2022) also considered the presence of glutamine (Q) at position 298 (296 considering deletions compared to A and B variants) as a key feature of D variant [77]. As shown in our study, C2 subvariant is completely different from D variant in this epitope, having 293E, 294T, 295D and 299K, the same as C1 variant, and 296N, the same as strain Tokyo_JP_1976. In addition, the presence of 297P in all representatives of C2 and C1/2 also brings them closer to strain Tokyo_JP_1976. The strains of the C2 and C1/2 subvariants have a HBGA binding site carrying the V444/442Y substitution. The presence of tyrosine (Y) in this position, as shown by Qian et al. (2018), allows new variants of the GII.17 genotype (C and D) to bind effectively to host receptors, unlike representatives of variants A and B [9]. Early strains carrying valine (V) in this position, which does not provide the necessary Van der Waals interaction with fucose, showed weak ability to bind host HBGA. It is interesting to note that of all the early GII.17 norovirus strains, only the Tokyo_JP_1976 strain also has tyrosine in this position. Previously, based on phylogenetic analysis of ORF2 gene sequences and amino acid substitutions in the VP1 protein, it was suggested that C variant, which emerged in 2012, could have originated from a virus similar to Tokyo_JP_1976 [86, 87], but for unknown reasons it was not detected for 37 years [9]. The results obtained in our study showed the return to active circulation of C variant, which had not circulated since January 2016. Data on amino acid substitutions in subvariant C2, which bring it even closer to the isolate Tokyo_JP_1976, indicate that the evolution of this phylogenetic lineage has continued in the last decade, has the character of convergence with the ancestral strain, and for four years (2017–2020) these processes were latent. According to the CDC website, genotype II.17 came in second place after GII.4 in the 2022–2023 epidemic season [88]. An increase in the share of genotype GII.17 in the Russian Federation was noted in 2022 and 2023 [89]. However, the emergence of this variant did not have a noticeable impact on the incidence of norovirus infection. Our calculation of the effective population size using the example of four genome regions confirmed the results of a similar analysis by Sang et al. (2018), obtained on the basis of the VP1 gene, which indicated the existence of an epidemic peak in 2014–2015 during the period of the appearance and spread of C and D variants [40]. But, as we have shown, the subsequent appearance of a new C2 subvariant and its circulation in a number of territories in 2021–2023 did not affect the effective population size. The analysis of partial genome sequences of noroviruses, including the overlap zone of non-structural and structural protein genes (RdRp-VP1), conducted in this study also showed the return to circulation in 2018 of the B variant of the GII.17 genotype, which had not been detected worldwide since 2009. Noroviruses with the GII.17/B capsid were detected in wastewater in South Africa in 2018–2022, in association with P7 and P17 polymerases [66]. The evolution of this variant, most likely in a latent form, continued on the African continent. More extensive nucleotide sequences of the genome of these noroviruses are needed for a detailed analysis. The obtained results provide additional information to characterize the evolutionary features of noroviruses of different genotypes. The concept of evolving and static genotypes of noroviruses was previously proposed [39]. Only GII.4 was classified as evolving, since the 1990s it had a change of epidemic variants every 2–3 years, with the previous variant being completely replaced by the next one, and by 2012 there were already eleven variants of this genotype. Static genotypes have a significantly smaller number of variants, and they circulate for many years, evolving in parallel. In particular, we confirmed this for the GII.6 genotype [90]. However, the GII.4 Sidney variant has been circulating in the world for more than 10 years since 2012 and evolves not due to mutations in the gene of the major structural protein, which lead to the formation of new epidemic variants, but due to the acquisition of other genes of non-structural proteins in the process of recombination [91]. New variants of the GII.4 genotype – Hong Kong2019[P31] and San-Francisco_2017[P31] – are antigenically different from previously circulating ones, have been identified in a number of countries in Europe and Asia, but have been circulating for more than 7 years without causing huge outbreaks. It is not yet clear, whether the pandemic potential of these variants will be realized or whether they are an example of suppressed circulation of minor norovirus variants [92]. Genotype GII.17, on the contrary, showed the features of an evolving genotype, when C variant was suddenly replaced by the dominant D variant in 2014–2015 shortly after its emergence in 2012. The latter circulated as a minor in the following years. However, the return to active circulation of C variant in 2022–2023 shown in our study, as well as the discovery of isolates of B variant after almost a 10 year break, indicates that the co-circulation of several independently evolving variants of the GII.17 genotype continues. This fact, in all probability, confirms its belonging to static genotypes and is consistent with the assumption that non-GII.4 noroviruses are characterized by linear evolution without substitution of variants. This evolution is due to minimal changes at the protein level due to a higher ratio of synonymous substitutions compared to non-synonymous ones, since VP1 of these viruses is under strong evolutionary constraints and cannot accumulate amino acid substitutions [87]. To better understand the spatiotemporal dynamics of this genotype, as well as to study evolutionary processes in the norovirus population in general, continuous surveillance of their circulation and retrospective studies based on archival samples are necessary. Declarations Author contributions NVE conceived and designed the experiments, wrote the manuscript. SVO and AEA performed the experiments. NVE and OVM analyzed the data. TAS and NAN revised the manuscript. 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J Gen Virol. 99(8):1027–1035. https://doi.org/10.1099/jgv.0.001088 Calicinet. https://www.cdc.gov/norovirus/reporting/calicinet/data.html Bykov R, Itani T, Starikova P, Skryabina S, Kilyachina A, Koltunov S, Romanov S, Semenov A (2024) Genetic Diversity and Phylogenetic Relationship of Human Norovirus Sequences Derived from Municipalities within the Sverdlovsk Region of Russia. Viruses 16(7):1001. https://doi.org/10.3390/v16071001 Epifanova, N.V. Genetic variants of norovirus of GII.6 genotype (2015) Mol. Genet. Microbiol. Virol. 30, 192–200. https://doi.org/10.3103/S0891416815040047 Parra GI (2019) Emergence of norovirus strains: A tale of two genes. Virus Evol 2019 5(2):vez048. https://doi.org/10.1093/ve/vez048 Tohma K, Landivar M, Ford-Siltz LA, Pilewski KA, Kendra JA, Niendorf S, Parra GI (2024). Antigenic Characterization of Novel Human Norovirus GII.4 Variants San Francisco 2017 and Hong Kong 2019. Emerg Infect Dis 30(5):1026–1029. https://doi.org/10.3201/eid3005.231694 Additional Declarations The authors declare no competing interests. Supplementary Files Sapplementarymatherial1.PhylogenetictreeMCCofGII.17NoVRdRpVP1fragment.pdf Sapplementary matherial 1. Phylogenetic tree MCC based on the nucleotide sequences of GII.17 NoV RdRp-VP1 fragment Sapplementarymatherial2.PhylogenetictreeMCCofGII.17NoVcompleteVP1gene.pdf Sapplementary matherial 2. Phylogenetic tree MCC based on the nucleotide sequences of GII.17 NoV complete VP1 gene Sapplementarymatherial3.PhylogenetictreeMCCofGII.17NoVcompleteVP2gene.pdf Sapplementary matherial 3. Phylogenetic tree MCC based on the nucleotide sequences of GII.17 NoV complete VP2 gene Sapplementarymatherial4.PhylogenetictreeMCCofGII.17NoVcompleteRdRpgene.pdf Sapplementary matherial 4. Phylogenetic tree MCC based on nucleotide sequences of GII.17 NoV complete RdRp gene Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5393670","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374257007,"identity":"0278e24d-5e32-4c62-a9d1-173689bdefd2","order_by":0,"name":"Epifanova N.V.","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYFACxoYPMNYDBgYJGWK0NM6AspgNgFp4iLIGpoVNAkgQ1sIv3dzY8HOHTWL/7B6zqhs1FjwM0mcM8GqRnHOwsbH3TFrijDtnzG7nHAM6jC8HvxaDG4ntD3jbDhsz3EhLu53DBtTCw4Nfi/2NxMbGv0At8kAtxTn/iNBiIJHY2Ay0Rc7gRvIx5tw2IrRI3DnY2CzbliZneCP5sHRunwQPGw9bAV4t/LPbHza+bbPhkQO68HPOtzo5fh7mDXi1MEigC7DhV49NyygYBaNgFIwCdAAAd1xBCuHrynYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7679-8029","institution":"I.N. 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Blokhina Nizhny Novgorod Research Institute of Epidemiology and Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Alekseeva","middleName":"","lastName":"A.E.","suffix":""},{"id":374257771,"identity":"0f16743a-1715-446a-a781-b83eb6e5b102","order_by":5,"name":"Novikova N.A.","email":"","orcid":"https://orcid.org/0000-0002-3710-6648","institution":"I.N. Blokhina Nizhny Novgorod Research Institute of Epidemiology and Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Novikova","middleName":"","lastName":"N.A.","suffix":""}],"badges":[],"createdAt":"2024-11-05 08:44:34","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5393670/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5393670/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68417478,"identity":"c4ac83ae-d7ab-4c4b-af87-4da1f8c70a72","added_by":"auto","created_at":"2024-11-07 05:27:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83434,"visible":true,"origin":"","legend":"\u003cp\u003eThe share of the GII.17P[17] genotype in the structure of the norovirus population in Nizhny Novgorod in different seasons of the studied period and the frequency of detection of noroviruses\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/be02b916582ebbde87e5e7ba.png"},{"id":68417748,"identity":"9f18b790-53c0-47a4-9547-a797a58cbfb9","added_by":"auto","created_at":"2024-11-07 05:35:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":143800,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree MCC based on the \u0026nbsp;nucleotide sequences of GII.17 NoV RdRp-VP1 fragment (size: 525 nt). A, B, D – variants of GII.17 NoV, C1, C2, C1/2 – subvariants of GII.17 NoV variant C.\u003c/p\u003e\n\u003cp\u003eBlue square – reference strain of C variant, black circles – isolates identified in Nizhny Novgorod, green triangles – isolates identified in other regions of Russia, red triangles – isolates of C2 subvariant identified in other countries\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/24574b9531b9bb196c36686e.png"},{"id":68417473,"identity":"dbeaf63f-f320-4c30-bcb4-3c007439012c","added_by":"auto","created_at":"2024-11-07 05:27:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112247,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree MCC based on the nucleotide sequences of GII.17 NoV complete VP1 gene (size:1632 nt). A, B, D – variants of GII.17 NoV, C1, C2, C1/2 – subvariants of GII.17 NoV variant C.\u003c/p\u003e\n\u003cp\u003eBlue square – reference strain of C variant, black circles – isolates identified in Nizhny Novgorod, red triangles – isolates of C2 subvariant identified in other countries\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/68a9977b360179da879ef12e.png"},{"id":68417469,"identity":"457eada8-d03c-4827-ab58-63a6351298d5","added_by":"auto","created_at":"2024-11-07 05:27:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":122577,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of differences in amino acid sequences of GII.17 NoV relative to the reference strain of variant C1 AB983218_C_Kawasaki323_JP_2014 (examined by applying the Poisson correction with MEGA 11). A, B, D – variants of GII.17 NoV, C1, C2, C1/2 – subvariants of GII.17 NoV variant C\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/eefb8c1e20c31cf962ae3210.png"},{"id":68417475,"identity":"4ffefbf1-5dc7-4b51-a5d2-dde92c791b51","added_by":"auto","created_at":"2024-11-07 05:27:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":60933,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid substitutions in the GII.17 NoV VP1 relative to the prototype C1 AB983218_C_Kawasaki323_JP_2014. Gray fill – deletions. Antigenic epitopes A, C, D, E are designated by analogy with the antigenic epitopes of norovirus genotype GII.4\u003c/p\u003e\n\u003cp\u003e* pink fill – HBGA binding sites\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/91c0e8a6258018b3d4ada495.png"},{"id":68417477,"identity":"4331c11c-9c30-40cd-a226-06d4e0d45996","added_by":"auto","created_at":"2024-11-07 05:27:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":250161,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial models of the VP1 protein P domain of GII.17[P17] NoV, side and top views (90° rotation): (a) based on the P domain of the Kawasaki323_JP_2014 strain (5F4M, PDB DOI: \u003ca href=\"https://doi.org/10.2210/pdb5F4M/pdb\"\u003ehttps://doi.org/10.2210/pdb5F4M/pdb\u003c/a\u003e, \u003ca href=\"https://www.rcsb.org/structure/5F4M\"\u003ehttps://www.rcsb.org/structure/5F4M\u003c/a\u003e), (b) based on the P domain of the Kawasaki308_JP_2015 strain (5F4O,PDB DOI: \u003ca href=\"https://doi.org/10.2210/pdb5F4O/pdb\"\u003ehttps://doi.org/10.2210/pdb5F4O/pdb\u003c/a\u003e, \u003ca href=\"https://www.rcsb.org/structure/5F4O\"\u003ehttps://www.rcsb.org/structure/5F4O\u003c/a\u003e ); the dark shade is subunit A, the light shade is subunit B. Deletionsare indicated in blue, red indicates a changein the polarity and/or chargeof amino acids, greenindicates the polarity and/ or charge of the amino acid has not changed\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/4a31b1b1c1a0e9973f50c0b8.png"},{"id":68420103,"identity":"18c51e68-52a3-4c4a-bf59-ab08e8f3d113","added_by":"auto","created_at":"2024-11-07 05:59:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":64791,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Phylogenetic tree MCC based on the nucleotide sequences of GII.17 NoV complete VP2 gene (size:735 nt). A, B, D – variants of GII.17 NoV, C1, C2, C1/2 – subvariants of GII.17 NoV variant C. (b) Amino acid substitutions in the GII.17 NoV VP2 relative to the C1 prototype AB983218_C_Kawasaki323_JP_2014\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/6e4876340119d44e26f4ddcc.png"},{"id":68417474,"identity":"11aebb7e-43c5-4fb8-a374-3c69e6a82a38","added_by":"auto","created_at":"2024-11-07 05:27:15","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":148794,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Phylogenetic tree MCC based on nucleotide sequences of GII.17 NoV complete RdRp gene (size:1380 nt). A, B, D – variants of GII.17 NoV, C1, C2, C1/2 – subvariants of GII.17 NoV variant C. (b) Amino acid substitutions in RdRp. (c) The spatial model of the RdRp GII.P17 NoV is constructed based on the RdRp GII.P4 NoV (4QPX, PDB DOI: \u003ca href=\"https://doi.org/10.2210/pdb4qpx/pdb\"\u003ehttps://doi.org/10.2210/pdb4qpx/pdb\u003c/a\u003e, \u003ca href=\"https://www.rcsb.org/structure/4QPX\"\u003ehttps://www.rcsb.org/structure/4QPX\u003c/a\u003e )\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/196e9c260a42355059c9a646.png"},{"id":68417472,"identity":"10697391-d4da-405b-adb3-2efb33683bcf","added_by":"auto","created_at":"2024-11-07 05:27:15","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":84915,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid substitutions in nonstructural proteins p48, NTPase, p22, VPg, Pro \u0026nbsp;of GII.17 NoV\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/045191696f1e2f0ed53a21de.png"},{"id":68417479,"identity":"2ea2d1ad-d853-40da-811d-1717abada17b","added_by":"auto","created_at":"2024-11-07 05:27:16","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":63505,"visible":true,"origin":"","legend":"\u003cp\u003eDemographic history of the fragment RdRp-VP1, VP1, VP2, RdRp genеs. The x-axis shows time in calendar years. The y-axis shows the effective population size of the virus, representing the number of genomes that are effective for the development of new infections. The line in the center represents median values within 95% of the target density range.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/19000f65376676b1f2458dd4.png"},{"id":68417464,"identity":"c9556c38-6eaa-4bda-9e51-700e250c1fe9","added_by":"auto","created_at":"2024-11-07 05:27:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":90030,"visible":true,"origin":"","legend":"\u003cp\u003eSapplementary matherial 1. Phylogenetic tree MCC based on the \u0026nbsp;nucleotide sequences of GII.17 NoV RdRp-VP1 fragment\u003c/p\u003e","description":"","filename":"Sapplementarymatherial1.PhylogenetictreeMCCofGII.17NoVRdRpVP1fragment.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/c525ece414a11a4505ffa6db.pdf"},{"id":68417744,"identity":"c84f0aef-a1b8-46f1-bdf9-b584469c47a0","added_by":"auto","created_at":"2024-11-07 05:35:14","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":48267,"visible":true,"origin":"","legend":"\u003cp\u003eSapplementary matherial 2. Phylogenetic tree MCC based on the nucleotide sequences of GII.17 NoV complete VP1 gene\u003c/p\u003e","description":"","filename":"Sapplementarymatherial2.PhylogenetictreeMCCofGII.17NoVcompleteVP1gene.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/56da4baef7cbe9c584a594ec.pdf"},{"id":68417747,"identity":"f28345b8-3ecd-4ff7-840d-1394a4958ab5","added_by":"auto","created_at":"2024-11-07 05:35:15","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":78497,"visible":true,"origin":"","legend":"\u003cp\u003eSapplementary matherial 3. Phylogenetic tree MCC based on the nucleotide sequences of GII.17 NoV complete VP2 gene\u003c/p\u003e","description":"","filename":"Sapplementarymatherial3.PhylogenetictreeMCCofGII.17NoVcompleteVP2gene.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/2461d17bc91569e1a7400be3.pdf"},{"id":68417476,"identity":"1356cec1-674a-4a9e-b59d-155a8a75b0e9","added_by":"auto","created_at":"2024-11-07 05:27:15","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":47677,"visible":true,"origin":"","legend":"\u003cp\u003eSapplementary matherial 4. Phylogenetic tree MCC based on nucleotide sequences of GII.17 NoV complete RdRp gene\u003c/p\u003e","description":"","filename":"Sapplementarymatherial4.PhylogenetictreeMCCofGII.17NoVcompleteRdRpgene.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5393670/v1/bae2dacc584a731a7d4500fd.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eRe-emergence and Spread of Norovirus Genotype Gii.17 Variant C in 2021-2023\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNoroviruses (NoVs) discovered more than half a century ago [1] belong to the family \u003cem\u003eCaliciviridae\u003c/em\u003e, genus \u003cem\u003eNorovirus\u003c/em\u003e (\u003cem\u003eViruses; Riboviria; Orthornavirae; Pisuviricota; Pisoniviricetes; Picornavirales; Caliciviridae\u003c/em\u003e).Currently, NoVs is associated with one fifth of all aсute gastroenteritis cases in the world and is estimated to cause over 200,000 deaths annually in developing countries [2]. NoVs is also the main etiological agent of outbreaks of non-bacterial gastroenteritis in kindergartens, schools, hospitals, camps, cruise ships, military units and nursing homes, affecting all age groups of the population [3].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eNoroviruses are small, non\u0026ndash;enveloped viruses (size: 38 nm), the capsid of which consists of 90 dimers of the major structural capsid protein VP1 and several molecules of the minor capsid protein VP2 [4]. The human norovirus genome is a linear single-stranded (+) RNA approximately 7.5\u0026ndash;7.7 thousand nucleotide bases (nt) in length with a polyadenylated 3'-end and a VPg peptide covalently linked to the 5- end, and is organized into three open reading frames (ORFs) [5]. ORF1 encodes a large polyprotein, the precursor of six nonstructural proteins (NS1/2 - NS7), including RNA-dependent RNA polymerase (RdRp, NS7), ORF2 overlaps at 14\u0026ndash;20 nt with ORF1 and is usually translated from subgenomic RNA [4].\u003c/p\u003e \u003cp\u003eORF2 encodes the major structural capsid protein VP1, which is subdivided into the N domain, the S domain forming the virion shell, and the P domain protruding above the shell surface [6]. The P domain consists of two relatively conservative subdomains P1-1 and P1-2, between which the highly variable P2 subdomain is located. P2 carries antigenic determinants and binding sites to host cell receptors (co-receptors) \u0026ndash; blood group antigens represented on intestinal enterocytes (histo-blood group antigens, HBGA) [7].\u003c/p\u003e \u003cp\u003eHBGA are complex fucose-containing glycans that define polymorphic human blood groups and play a critical role in susceptibility to noroviruses [8, 9]. ORF3 encodes the minor structural capsid protein VP2 and overlaps with ORF2 at 1 nt [4]. VP2 is located inside the viral particle and participates in capsid assembly and genome encapsulation [10, 11].\u003c/p\u003e \u003cp\u003eAt the junction of the reading frames encoding non-structural and structural proteins, there is a \u0026ldquo;hot spot\u0026rdquo; where recombinations often occur in the norovirus genome, so a dual nomenclature is currently adopted that takes into account norovirus genotypes in two reading frames. The ORF1 region encoding RNA polymerase and the ORF2 region encoding the N/S domain and P domain of the capsid protein VP1 are most frequently used for typing [12].\u003c/p\u003e \u003cp\u003eAccording to the classification presented in 2019, NoVs divided into ten genogroups (GI-GX). Based on the analysis of the amino acid sequence of the main capsid protein VP1, 48 genotypes are isolated, based on the analysis of the nucleotide diversity in the RdRp region, noroviruses are divided into 60 P-types [13]. Some genotypes and P-types, for which single nucleotide sequences still exist, are not assigned numbers, they are called NA. Mutations that cause changes, insertions or deletions of amino acids in VP1 change the properties of the virus, affect the communication and formation of the immune system and lead to the appearance of new antigenic variants of NoVs [14, 15].\u003c/p\u003e \u003cp\u003eWorldwide, genotype GII.4 is responsible for most outbreaks and sporadic cases of gastroenteritis associated with NoVs since the 1990s [16]. The circulation of noroviruses of other genotypes periodically intensifies, acquiring epidemic distribution [17, 18, 19].\u003c/p\u003e \u003cp\u003eGII.17 viruses have been detected in the world for more than four decades. The earliest are noroviruses identified in the study of archival samples of copromaterial collected in 1978 in French Guiana [20] and in 1976 and 1982 in Japan [21]. Until 2012, cases of GII.17 registration worldwide were rare [22]. One of the few exceptions was the identification of the GII.17 genotype in 76% of noroviruses detected in 2012\u0026ndash;2013 in surface waters of rivers in rural and urban areas of Kenya [23].\u003c/p\u003e \u003cp\u003eHowever, in the winter of 2014\u0026ndash;2015, the number of outbreaks caused by the GII.17 genotype increased sharply in Southern China [24, 25], Hong Kong [26], Japan [27] and South Korea [28]. An outbreak outside Asia caused by this genotype was reported in Romania in October-December 2015 [29]. In Russia, aquatic outbreaks of norovirus infection caused by GII.17 were described in the Khabarovsk Krai in March-June 2015 [30] and in the Republic of North Ossetia-Alania in June 2015 [31]. GII.17 norovirus has not only caused outbreaks but has also been sporadically detected in China (Shanghai, Taiwan) [32, 33], the United States of America [34], and Latin America [35, 36, 37, 38].\u003c/p\u003e \u003cp\u003eCurrently, GII.17 noroviruses are divided into four variants \u0026ndash; A, B, C and D [39]. A and B variants include noroviruses detected in isolated cases since the 70s of the last century in different countries of the world and having a polymerase gene with specificity P4, P3, P13, P16, P31 [22]. C variant, which appeared in 2012, had a polymerase type that had not previously been detected in association with other VP1 genotypes, and was designated P17 [27].\u003c/p\u003e \u003cp\u003eSomewhat later, D variant with the same P17 polymerase emerged and has spread widely around the world since 2014\u0026ndash;2015, soon completely replacing C variant [40]. The new GII.17 variants (C and D) were found to have stronger HBGA binding ability than the previous variants (A and B) [9, 41, 42].\u003c/p\u003e \u003cp\u003eIt was assumed that GII.17 norovirus may have pandemic potential and replace genotype GII.4, the epidemic variants of which have dominated the world since the mid-1990s [22]. However, later the intensity of GII.17 circulation decreased everywhere, and only in 2022\u0026ndash;2023 we noticed an increase in its share in the spectrum of norovirus genotypes circulating in Nizhny Novgorod.\u003c/p\u003e \u003cp\u003eThe aim of the work is the molecular genetic characteristics of GII.17 noroviruses, identified in Nizhny Novgorod, in comparison with the dynamics of circulation of noroviruses of this genotype in the world.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eSamples under study.\u003c/b\u003e The study used fecal samples from children hospitalized with symptoms of acute intestinal infection in the children's infectious diseases hospital of Nizhny Novgorod in the period 2014\u0026ndash;2023. RNA was extracted and precipitated using a RIBO-prep reagent kit (Central Research Institute of Epidemiology [CRIE], Russia). Norovirus was detected using a real-time PCR diagnostic kit (AmpliSens Rotavirus/Norovirus/Astrovirus-FL and AmpliSens OKI viro-screen-FL\", CRIE). Virus cDNA for genotyping and amplification before sequencing was obtained using a REVERTA-L kit [CRIE].\u003c/p\u003e \u003cp\u003e \u003cb\u003eSequencing of the norovirus genome\u003c/b\u003e. To determine the genotype of noroviruses, cDNA sequencing was performed on a 570-nt region of the viral genome, including the overlap of open reading frames for nonstructural and structural proteins and encoding the following regions: the C-terminal region of RNA-dependent RNA polymerase and the N-terminal region of the S-domain of the capsid protein VP1 (RdRp-VP1).\u003c/p\u003e \u003cp\u003eThe method based on a combination of two pairs of previously published primers [43, 44], was proposed by Cannon J.L. et al. [45]. For cDNA amplification, we used reagents manufactured by Sileks Ltd., Evrogen Ltd. Syntol Ltd. (Russia) and primers synthesized in Syntol Ltd. and Evrogen Ltd. (Russia).\u003c/p\u003e \u003cp\u003eTo determine the complete sequence of the structural protein gene VP1 of GII.17 norovirus, amplification of three overlapping fragments was carried out using three pairs of specially selected primers (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eFragments for sequencing were purified using DNA fragment purification kits from gel (FractalBio Ltd., Russia). Determination of the primary structure of cDNA fragments of the norovirus genome was carried out by the Sanger method in automatic mode on genetic analyzers Beckman Coulter GenomeLab GeXP (Beckman Coulter, USA), Nanofor-05 (Institute for Analytical Instrumentation, Russia) using DTCS Quick Start Kit (Beckman Coulter, USA), BigDye Terminator v3.1 (Applied Biosystems, Thermo Fisher Scientific, USA), GenSek (Syntol, Ltd., Russia).\u003c/p\u003e \u003cp\u003eWhole genome sequencing was performed on the Illumina platform for one norovirus isolate. To obtain norovirus whole genome cDNA, reverse transcription was performed in the presence of the Tx30SXN primer [46], specific to the 3'-end of the viral genome, using the RNAscribe RT Reverse Transcriptase reagent kit (Biolabmix Ltd., Russia). Amplification of whole-genome cDNA fragments was performed in the presence of Tx30SXN and GII_1\u0026ndash;35 primers specific to the 3'- and 5'-ends of the viral genome [39] using the BioMaster LR HS-PCR-Color-2x kit (Biolabmix Ltd., Russia). The concentration of viral cDNA was assessed using a Qubit 2.0 fluorimeter (Invitrogen, Austria) and the Spectra Q HS kit for quantitative DNA determination (Raissol, Russia). Preparation of cDNA libraries for sequencing was performed using the ShotGun \"SG GM\" kit and a set of indexed primers for dual barcoding for Illumina sequencers (Raissol, Russia). Sequencing was performed on an iSeq100 instrument (Illumina, USA). The obtained archives of viral genome sequence readings were processed using the Unipro UGENE software [47], version 42.0. The GII.17 NoV whole genome from the GenBank database [48, 49] were used as a reference genome. As a result of combining short reads, a consensus sequence of the norovirus genome with coverage from 10x to 300x was obtained.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNorovirus genotyping.\u003c/b\u003e The resulting sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) [50, 51], as well as using web services for automatic norovirus genotyping Norovirus Genotyping Tool Version 2.0 [52, 53], Calicivirus typing tool [54, 55], NoroNetRus [56, 57].\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenBank numbers\u003c/b\u003e. The partial nucleotide sequences obtained in this study were registered in the international GenBank database with accession numbers MK033836-MK033839, MN542836-MN542838, ON854092-ON854113, ON854111, ON854112, OR475165-OR475194, the full genome sequence \u0026ndash; with the accession number OP712199.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analysis.\u003c/b\u003e A preliminary phylogenetic analysis of the nucleotide sequences we established was performed in the MEGA 11 program using the Maximum Likelihood method, using the Tamura-Nei substitution model [58] with five reference strains of noroviruses of the GII.17 genotype: KC597139, MW305610, AB983218, LC037415, AB684681.\u003c/p\u003e \u003cp\u003eWe then compiled a set of related sequences of norovirus genotype GII.17 from the GenBank database. The search for related sequences was performed using the BLAST. Nucleotide sequence alignment was performed using MEGA software, version 11 [58].\u003c/p\u003e \u003cp\u003eA 525 nt region of the RdR-VP1 viral genome was used for the analysis. A total of 485 norovirus sequences identified from 1976 to 2023 in 34 countries were downloaded from the Genbank database as of March 2024. Sequences with high homology, identical time and place of isolation were removed from the alignment. As a result, 377 sequences were included in the file for analysis, including 56 obtained in this study.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis based on sequences of complete genes VP1 (238 sequences including 15 obtained in this study), VP2 (216 sequences, including one of the Nizhny Novgorod isolate) and RdRp (215 sequences including one of the Nizhny Novgorod isolate) was carried out separately.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis and effective population size (EPS) calculation were performed using the Bayesian Skyline model [59] with the BEAUti 1.10.4 and BEAST v 1.10.4 [60] software packages. Rates of nucleotide substitution per sites and time to the most recent common ancestor (tMRCA) were estimated using Bayesian Markov chain Monte Carlo (MCMC). MCMC length was 500\u0026nbsp;million to 1\u0026nbsp;billion generations depending on the region (gene) analyzed to ensure effective sample size (ESS) values\u0026thinsp;\u0026gt;\u0026thinsp;200. The nucleotide substitution process was estimated using the Hasegawa\u0026ndash;Kishino\u0026ndash;Yano (HKY) model. Runs were performed under relaxed (uncorrelated lognormal) clock model. Visualization of population dynamics and analysis of output MCMC files were estimated using Tracer v1.7.1.1. [61]. The Maximum Clade Credibility (MCC) tree was summarized from the posterior distribution of trees using TreeAnnotator v1.10.4 included in the BEAST package and visualized and annotated using FigTree v1.4.4 [62].\u003c/p\u003e \u003cp\u003eThe amino acid variations within and between clades observed in the tree for VP1 were examined by applying the Poisson correction with MEGA 11.\u003c/p\u003e \u003cp\u003e \u003cb\u003eModeling the structure of the VP1 P-domain and RdRp.\u003c/b\u003e Molecular graphics were performed using UCSF ChimeraX [63, 64, 65]. Spatial models of the VP1 protein P domain of GII.17[P17] NoV are constructed based on the P domain of the Kawasaki323_JP_2014 strain, (5F4M, accession code in the Protein Databank \u0026ndash; PDB DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2210/pdb5F4M/pdb\u003c/span\u003e\u003cspan address=\"10.2210/pdb5F4M/pdb\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/structure/5F4M\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/structure/5F4M\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and on the P domain of the Kawasaki308_JP_2015 strain, (5F4O, PDB DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2210/pdb5F4O/pdb\u003c/span\u003e\u003cspan address=\"10.2210/pdb5F4O/pdb\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/structure/5F4O\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/structure/5F4O\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ). The spatial model of the RdRp GII.P17 NoV is constructed based on the RdRp GII.P4 NoV, (4QPX, PDB DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2210/pdb4qpx/pdb\u003c/span\u003e\u003cspan address=\"10.2210/pdb4qpx/pdb\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/structure/4QPX\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/structure/4QPX\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFrom July 2014 to June 2023, fecal samples from 14,180 children hospitalized in a pediatric infectious diseases hospital were tested for noroviruses. Noroviruses were detected in 2,438 children (17.19% of cases). The genotype was determined for 691 isolates, genotype GII.17 was identified in 70 cases (10.13%).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe share contribution of genotype GII.17 to the population structure of noroviruses in the period 2014\u0026ndash;2023 in Nizhny Novgorod\u003c/b\u003e. NoV GII.17 was first detected in Nizhny Novgorod at the end of the 2014\u0026ndash;2015 season (in June 2015) and in the 2015\u0026ndash;2016 season, it took second place in the spectrum of norovirus genotypes after GII.4, accounting for 28.1% of the number of typed isolates. Subsequently, its share fluctuated from 3.0 to 10.6% in different epidemic seasons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe sharp decline in the detection of GII.17 norovirus in the second half of 2016 and its subsequent detection in no more than 10\u0026ndash;11% of cases suggested that GII.17 norovirus had largely exhausted its epidemic potential and would continue to circulate at low frequency as a minor genotype.\u003c/p\u003e \u003cp\u003eHowever, in the 2022\u0026ndash;2023 season, an increase in the share of GII.17[P17] in the spectrum of detected genotypes to 36.1% was observed. Moreover, this was not accompanied by an increase in the frequency of detection of noroviruses; on the contrary, the indicator even decreased slightly compared to the previous season (13.8% \u0026minus;\u0026thinsp;9.9%, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To identify the characteristics of new strains of this genotype, a philodynamic analysis was conducted based on the nucleotide sequences of the GII.17 norovirus genome circulating in Nizhny Novgorod, as well as those extracted from the GenBank database.\u003c/p\u003e\n\u003ch3\u003eAnalysis of RdRp-VP1 fragment\u003c/h3\u003e\n\u003cp\u003eFor four isolates of GII.17 norovirus, identified in Nizhny Novgorod in 2015\u0026ndash;2016, partial nucleotide sequences of the N/S domain of the VP1 gene were obtained, and for 56 isolates identified in 2017\u0026ndash;2023, sequences of the region including the overlap zone of open reading frames encoding RNA-dependent RNA polymerase and the VP1 capsid protein (RdRp-VP1 fragment ) were obtained. The latter were used to construct a phylogenetic tree with reference sequences of A (KC597139_A_C142_GF_1978), B (MW305610_B_4522_AR_2005), C (AB983218_C_Kawasaki323_JP_2014) and D (LC037415_D_Kawasaki308_JP_2015) variants, as well as the earliest isolate of genotype GII.17, identified in an archival sample of 1976 from Tokyo (Japan), carrying P3 polymerase and not assigned to any variant (AB684681_ 27\u0026thinsp;\u0026minus;\u0026thinsp;3/Tokyo_JP_1976).\u003c/p\u003e \u003cp\u003ePhylogenetic analysis of the studied region (size: 525 nt) showed that the Nizhny Novgorod NoVs identified in 2017\u0026ndash;2022 belonged to D variant together with the reference strain Kawasaki308_JP_2015. The Nizhny Novgorod isolates of 2021\u0026ndash;2023 formed a reliable cluster with the strain Kawasaki323_JP_2014 \u0026ndash; the prototype of C variant, although they formed a separate branch within this cluster, which we designated \" C2 subvariant \" in contrast to the original strain, which in this context we will call the \"C1 subvariant\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Isolate 27\u0026thinsp;\u0026minus;\u0026thinsp;3/Tokyo_JP_1976 turned out to be closer in this region to representatives of C and D variants than to A and B variants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eComparison of aligned fragments showed that C2 subvariant carries nucleotide substitutions that distinguish it from both the prototype C variant and the prototype D variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These substitutions allow identifying a new subvariant even when sequencing shorter genome regions. Thus, four Nizhny Novgorod isolates of 2015\u0026ndash;2016, for which we obtained only regions of the N/S domain of the VP1 gene, according to this analysis belonged to D variant, since they had characteristic substitutions C379T, C473T, C479T.\u003c/p\u003e \u003cp\u003eTo clarify the origin of the new GII.17 subvariant, the sample was significantly expanded and analyzed using the Bayesian phylodynamic approach. The phylogenetic tree constructed on the basis of 377 sequences, the common ancestor for all analyzed sequences existed in 1848 (Supplementary material 1, Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eVariant A includes 12 sequences of noroviruses circulated in 2002\u0026ndash;2019 in the USA, Great Britain, Guatemala, Argentina, China, Paraguay, Japan, and South Africa and carried polymerase genes with P16 and P31 specificity. The prototype strain KC597139_C142_GF, identified in 1978 in French Guiana, also belongs to this variant. It should be noted that the polymerase genotype of this strain was previously annotated as P4 [22], but in automated norovirus genotyping systems it is currently defined as P12.\u003c/p\u003e \u003cp\u003eVariant B includes 14 sequences of noroviruses with P13 polymerase circulated in 2004\u0026ndash;2009 in France, Cameroon, Argentina, Peru, China, Russia (Chelyabinsk), as well as 7 sequences of noroviruses detected in 2019\u0026ndash;2020 in wastewater in Pretoria (South Africa), carrying P7 and P17 polymerase [66], and one sequence of noroviruses with P17 from Ecuador (2019). It is worth noting that isolate DQ438972_B_Katrina_US_2005, often used as a reference strain for B variant, is not included in this analysis because its polymerase gene sequence has not been determined.\u003c/p\u003e \u003cp\u003eAn intermediate position between B and C variants is occupied by sequences of three isolates from Japan (two from 1976 with P3 and P13 [21], one from 1982 [20], whose polymerase is closest to P40), one isolate from Tunisia (1977) with polymerase P13, one isolate of norovirus with polymerase P3, identified in Saudi Arabia in 1990, and one isolate from Argentina (2015) with polymerase PNA6 [67].\u003c/p\u003e \u003cp\u003eThe divergence of C and D variants dates back to 2005 (Table\u0026nbsp;2). Variant C (91 sequences) in this tree is represented by a single branch with tMRCA, which existed in 2010. At the base of this branch is subvariant C1, from which subvariant C2 evolved (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e supplementary materials). C1 subvariant includes noroviruses with P17 polymerase which circulated in 2012\u0026ndash;2015 in China, Brazil, the Netherlands, Australia, India, South Korea, France, Canada, Japan (including the earliest isolate LC433694_OC12019_JP_2012, detected in Osaka in March 2012 [68] and the reference strain AB983218_C_Kawasaki323_JP_2014), as well as the only 2016 strain from the UK [69].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eC2 subvariant with tMRCA that existed in 2020 includes sequences of 33 Nizhny Novgorod isolates (including 1 from 2021, 22 from 2022, and 11 from 2023), one isolate from the USA (2022), 3 from Romania (2021) [70], 13 from Brazil (2023) [71], and 3 isolates from Russia (two from Khabarovsk, 2022, and one from Kamensk-Uralsky, Sverdlovsk Region, 2023). It is noteworthy that we did not find C variant strains circulating in the period 2017\u0026ndash;2020 in the GenBank database.\u003c/p\u003e \u003cp\u003eAlignment of partial sequences available in GenBank encoding the N/S domain of the VP1 protein of noroviruses circulating in other regions of Russia, which were not included in this analysis, and analysis of characteristic nucleotide substitutions showed that 18 isolates obtained in the Sverdlovsk region in 2022\u0026ndash;2023 (OP862435-OP862440, OR399139, OR399140, OR447705-OR447707, OR717543-OR717547, OR726224) also belong to the C2 subvariant (data not shown).\u003c/p\u003e \u003cp\u003eVariant D (reference strain LC037415_D_Kawasaki308_JP_2015) includes the majority of sequences (254) in the phylogenetic tree we constructed, including sequences of twenty-three Nizhny Novgorod norovirus isolates of 2017\u0026ndash;2022 and two isolates identified in Amursk, Khabarovsk Krai, in 2022 (OR775312, OR775313) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, supplementary materials). Noroviruses of variant D, whose tMRCA according to the topology of this tree existed in 2010, circulated widely in the world in 2014\u0026ndash;2022. The vast majority of them had polymerase P17. The exceptions were two isolates with polymerase P25 (France 2014, Netherlands 2016), 8 isolates from South Africa 2018\u0026ndash;2020, including 6 with polymerase P7, one with P16 and one with P33 (Supplementary material 1).\u003c/p\u003e \u003cp\u003eThe overall mutation accumulation rate for the GII.17 genotype based on the analyzed genomic region was 1.78E-3 (1.09E-3 ‒ 2.55E-3) substitutions per site per year (Table\u0026nbsp;2). Individual clusters evolved at a comparable rate, with this figure being slightly higher for variant D (5.38E-3).\u003c/p\u003e\n\u003ch3\u003eORF2 analysis\u003c/h3\u003e\n\u003cp\u003eFor 15 Nizhny Novgorod isolates, complete nucleotide sequences of ORF2 encoding the major structural protein VP1 were determined. These sequences as well as the ORF2 sequences of the strain for which the whole genome sequence was obtained (OP712199_755_NN_RU_2022), were included in the sample for phylogenetic analysis of 238 sequences.\u003c/p\u003e \u003cp\u003eThe topology of the obtained phylogenetic tree is generally similar to that for the RdR-VP1 fragment (see Supplementary materials 2). Clusters of A (12 sequences), B (11 sequences), D (174 sequences, including three Nizhny Novgorod isolates from 2020) variants are clearly distinguished.\u003c/p\u003e \u003cp\u003eStrains AB684681_P3_27\u0026thinsp;\u0026minus;\u0026thinsp;3/Tokyo_JP_1976 and MW305625_PNA6_ Arg13099_AR_2015 also occupy an intermediate position between B and C variants. The common ancestor for all analyzed VP1 gene sequences existed in 1806 (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eHowever, unlike the tree for the RdRp-VP1 fragment, in this phylogenetic tree C variant (41 sequences) is heterogeneous (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The strain MH218689_231_GB_2016, as well as the strains MK907790_G19_020_FR_2014 and MW661253_BMH15-067_Ca_2015 separated from C1 subvariant, which had tRMCA in 2009. The latter in this tree occupy an intermediate position between C1 and C2 variants and are designated \"C1/2 subvariant\", their common ancestor with isolates of C2 subvariant existed in 2011. C2 subvariant is formed by 13 Nizhny Novgorod isolates from 2021\u0026ndash;2023, 3 isolates from Romania (2021) and one from the USA (2022), whose tMRCA dates back to 2017. The evolution rate of D variant and C2 subvariant based on the VP1 gene (5.78E-3 and 4.7E-3, respectively) significantly exceeded this indicator for the genotype as a whole (1.90E-3) (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the obtained alignment of nucleotide sequences, the derived amino acid sequences were obtained by translation in the MEGA program. For the VP1 protein, the differences between the amino acid sequences of representatives of different variants and subvariants were determined using the Poisson correction.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows a histogram of the distribution of differences, expressed as a percentage, relative to the reference strain of C variant, C1 subvariant, AB983218_C_Kawasaki323_JP_2014. It can be seen that within C1 subvariant the indicator ranges from 0 to 0.19% (on average 0.16%), for isolates of C2 subvariant the range varies from 2.07 to 2.64% (on average 2.26%). For strains MK907790_G19_020_FR_2014 and MW661253_BMH15-067_Ca_2015, which, in our opinion, are an intermediate link between C1 and C2 subvariants (C1/2 subvariant), the differences from the prototype strain are 1.68% and 1.87%, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe differences with representatives of D variant range from 4.18 to 4.76 (on average 4.42%), A variant \u0026ndash; from 10.20 to 11.45 (on average 10.69%), B variant \u0026ndash; from 12.48 to 12.90 (on average 12.76%)\u003c/p\u003e \u003cp\u003eTaking into account the fact that the criterion for defining a new variant adopted for the GII.4 genotype (maximum amino acid variability within a variant is 4.1% [40]) was adapted for GII.17, isolates that differ from the prototype strain C by only 2.26% should be classified as belonging to the same variant.\u003c/p\u003e \u003cp\u003eWhen aligning the amino acid sequences of representatives of all four variants, it was found that the new subvariant, compared to A and B variants, has deletions at positions 295, 296, and 385 and an insertion/substitution at 346/346A, as do representatives of C and D variants (data not shown). It should be noted that in publications by different authors there are some shifts in the position of deletions and insertions depending on the alignment performed. These positions are indicated in accordance with the alignment presented in the article by Sang et al. (2018) [39].\u003c/p\u003e \u003cp\u003eAlignment of the new C2 subvariant sequences with only the VP1 amino acid sequences of C and D variants revealed differences with C1 variant at eleven amino acids, with no insertions or deletions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). One substitution is located in the S domain, three in the P1 subdomain, and seven in the P2 subdomain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere are 30 differences between the representatives of the new subvariant and the D variant, including two deletions at positions 373 and 397, and 28 amino acid substitutions, of which two are located in the S domain, four in the P1 subdomain, and 25 in the P2 subdomain (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe following substitutions are characteristic of C2: 296N, 297P, 361R, 372K, 385/384R, 411/409V, 433/431L, 437/435S (the positions are indicated for D variant and subvariant C2, respectively, taking into account deletions). The substitutions 296N, 297P and 385/384R are also present in the strain AB684681_P3_Tokyo_JP_1976, which, according to the phylogenetic analysis, is close to C and D variants, the substitution 411/409V was found in one strain of C1 subvariant - OP205532_R02-06_NL_2015 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The 433L substitution is present in two strains D variant from USA (MT344181_118-3_US_2015 and MT371769_PNA_147-2_US_2015) (data not shown). At position 377/376, both the amino acid N, found in most strains of the C1 subvariant, and the D, characteristic of the D variant, are present in different representatives of the C2 subvariant. Only the 361R, 372K, and 437/435S substitutions are unique to C2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe strains of subvariant C1/2 occupy an intermediate position, having amino acids similar to variant C1 in three cases (334V, 433/431F, 437/435F), in six cases \u0026ndash; to the new subvariant C2 (144I, 296N, 297P, 385/384R, 411/409V, 449/447I), in one case \u0026ndash; as in individual representatives of C1, C2 and all representatives of variant D (377/376D). The strain. The only unique substitution for C1/2 subvariant is 361K (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the positions of amino acids that make up the conformational epitopes identified in GII.4 noroviruses \u0026ndash; A, C, D, E, the location of which is mapped by analogy with those identified for GII.4 ([14, 72, 73], and also taking into account some experimental data obtained for GII.17 [74, 75, 76, 77]. It is evident that the amino acid substitutions characteristic of C2 subvariant are found in epitopes A (296, 297) and E (411/409) and may cause differences in antigenicity from both C1 subvariant and D variant.\u003c/p\u003e \u003cp\u003eFive amino acid residues that typically interact with α-1,2-fucose of HBGA (348/348T, 349/349R, 378/377D, 443/441G, 444/442Y for D and C variants, respectively, indicated by asterisks and pink shading in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) were highly conserved and identical for all representatives of variants C and D, and were also present in strain AB684681_P3_Tokyo_JP_1976.\u003c/p\u003e \u003cp\u003eThe amino acid substitutions we identified for VP1 of C2 NoV subvariant were plotted on the following structural models: P-domain of GII.17 norovirus Kawasaki323 (C1 variant), and P-domain of GII.17 norovirus Kawasaki308 (D variant) [74]. The resulting spatial models clearly demonstrate a relatively small number of amino acid substitutions compared to subvariant C1 and multiple substitutions compared to D variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eORF3 analysis\u003c/h3\u003e\n\u003cp\u003eFor one Nizhny Novgorod isolate, a whole genome nucleotide sequence was obtained, which allowed it to be included in the analysis of ORF3, encoding the minor capsid protein VP2. A total of 216 sequences were included in the analysis.\u003c/p\u003e \u003cp\u003eThe topology of the obtained tree is also generally similar to the trees for the VP1- RdRp region and for the VP1 gene (see Supplementary material 3). The common ancestor for all analyzed VP2 gene sequences existed in 1833 (Table\u0026nbsp;3). Differences are again observed for C variant, which is represented by two branches (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Strain MH218689_231_GB_2016 did not form a separate lineage, but entered the branch of C1 subvariant with the closest common ancestor in 2010. The second branch again split into two subbranches, one of which contains isolates from France in 2014 and from Canada in 2015 (MK907790_G19_020_FR_2014 and MW661253_BMH15-067_Ca_2015, C1/2 subvariant), and the other - C2 subvariant, which includes, as well as the VP1 tree, one Nizhny Novgorod isolate of 2022, three isolates from Romania (2021) and one from the USA (2022). The common ancestor of this branch existed in 2012, and C2 subvariant \u0026ndash; in 2018 (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe evolution rate for the genotype as a whole based on the VP2 gene (2.72E-3) was slightly higher than based on other regions of the genome, and for D variant, C2 subvariant, and also for C and D clusters as a whole (5.97E-3, 4.65E-3, and 7.01E-3, respectively) it significantly exceeded this indicator (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eAccording to the amino acid sequence of the VP2 protein, representatives of C2 subvariant have 9 substitutions compared to C1 variant, and 12 compared to D variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). At positions 145 and 161 of the new subvariant, the presence of amino acids characteristic of some isolates of variant B (S) is observed, and at position 243 \u0026ndash; for variants A and B (R), at position 146 \u0026ndash; for the reference strain of variant A (S) (data not shown). In a number of positions, representatives of the new subvariant have unique substitutions \u0026ndash; 30S, 58N, 145G. Substitutions 140S and 186R are present only in strains from Romania (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb).\u003c/p\u003e\n\u003ch3\u003eORF1 analysis\u003c/h3\u003e\n\u003cp\u003eThe most important nonstructural protein encoded by ORF1 is RNA-dependent RNA polymerase (RdRp). The complete polymerase gene was analyzed for 215 sequences (Supplementary materials 4) (including one from Nizhny Novgorod). The sequences are divided by the type specificity of the polymerase and grouped by variants: P12, P16, P31 \u0026ndash;A variant, P13 \u0026ndash; B variant, P17 \u0026ndash; C and D variants. Sequences with the type specificity P3, P25, PNA6 occupy an intermediate position.\u003c/p\u003e \u003cp\u003eThe common ancestor for the polymerase genes existed 100 years earlier (in 1733) than for the structural protein genes, which is explained by the different type affiliation of the analyzed genes, compared to the single type affiliation of the VP1 and VP2 genes (GII.17) (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eVariant C for the polymerase gene is represented by two branches - the first is subvariant C1, the second branch again split into two subbranches, which are formed by the same isolates as in the trees for ORF2 and ORF3 - subvariant C1/2 (isolates from France in 2014 and from Canada in 2015) and subvariant C2 (1 isolate from Nizhny Novgorod in 2022, 3 isolates from Romania (2021) and one from the USA (2022)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). Moreover, the time of existence of the closest common ancestors for the clusters presented in the table (except for the genotype as a whole) practically coincides with the genes of structural proteins (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe evolution rate of D variant and C2 subvariant based on the RdRp gene (3.99E-3 and 3.17 E3, respectively) slightly exceeded this indicator for the genotype as a whole (1.70 E-3) (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eAnalysis of amino acid substitutions in the P17 polymerase sequence in comparison with Kawasaki323 showed that most representatives of C variant (including the new C2 subvariant) have amino acid L at position 206, unlike the prototype strain, which carries F at this position. F at position 206 is also present in all representatives of D variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). At position 88, strain Kawasaki 323 has R, just like of subvariant C1/2 strains, unlike all other representatives of C and D variants.\u003c/p\u003e \u003cp\u003eThe five analyzed isolates of the new subvariant, compared to the representatives of C1 and D variants, are characterized by the substitutions R49K, N79K, N81S, Q102E, I215V (the last one \u0026ndash; with the exception of the strain from the USA) and A293V (in the strain from the USA \u0026ndash; A293T). Similar substitutions at positions 79 and 102 are present in two isolates of C1/2 subvariant, while the substitution V395I was found only in the sequences of the Romanian isolates.\u003c/p\u003e \u003cp\u003eThe amino acid substitutions of Rdp GII.17 norovirus C2 subvariant were mapped onto the structural model of GII.P4 norovirus polymerase [78] (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec). It is evident that the substitutions at positions 206 and 215 are located at the hydrophobic cleft near the active center of the polymerase.\u003c/p\u003e \u003cp\u003eWhen aligning the deduced amino acid sequences encoding other nonstructural proteins, substitutions characteristic of the C2 subvariant were noted in the region of protein P48 (56T, 58R, 85A, 195N, 198A, 276V, 281K, 286M), NTPase (453S, 464V), P22 (754T, 794 T/A, 830T) and VPg (966R) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). In the protease region, only the I1056M substitution is unique, present in two isolates of the C1/2 subvariant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDemographic analysis Skyplot\u003c/h2\u003e \u003cp\u003eDemographic inference using the Bayesian skyline plot (BSP) was performed on samples for the RdRp-VP1 region and the complete VP1, VP2, RdRp genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). All four plots show a steady decline in the effective population size (EPS), which became apparent after 2005. Around 2015, a peak in EPS growth was noted, which for structural and non-structural proteins was shifted by 1 year, and for the RdRp-VP1 region it was extended by two years, after which this value decreased again and a tendency to reach a plateau was observed. The emergence of new C 1/2 and C2 subvariants did not affect the EPS value. Indeed, we did not observe an increase in the frequency of norovirus detection in the 2022\u0026ndash;2023 season; on the contrary, there was a slight decrease in this indicator compared to the previous season (from 13.8\u0026ndash;9.9%, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGII.17 noroviruses, which caused an increase in the incidence in East Asia in 2014\u0026ndash;2015 and subsequently spread throughout the world, have been detected in a relatively small percentage of cases in recent years [79, 80, 81]. However, during the 2022\u0026ndash;2023 season, when monitoring the circulation of noroviruses in Nizhny Novgorod, a significant increase in the share of this genotype in the genetic spectrum of the norovirus population was observed.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis of the region of the norovirus genome, including the zone of overlapping reading frames encoding non-structural and structural proteins (RdRp-VP1), showed that 377 analyzed sequences were divided into four clusters corresponding to A, B, C and D variants. At the same time, the Nizhny Novgorod isolates of 2018\u0026ndash;2022 belong to D variant, which has been widespread in the world in recent years.\u003c/p\u003e \u003cp\u003eC variant, in our opinion, is divided into two subvariants. We identified C1 subvariant, which, as phylogenetic analysis showed, was detected not only in Asian countries, but was present in Europe and Latin America in 2013\u0026ndash;2016 [69, 82, 83] and was not detected in the world after 2016.\u003c/p\u003e \u003cp\u003eAnother subvariant, which included the Nizhny Novgorod isolates of 2023, as well as some isolates of 2022 and one of 2021, cluster with noroviruses from Romania (2021), the USA (2022) and Brazil (2023). Based on the topology of the phylogenetic tree constructed using the VP1-RdRp fragment, this group can be considered as a \"derivative\" of noroviruses of the C1 variant circulating in 2012\u0026ndash;2016, and represent a new GII.17 C2 subvariant.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis of the VP1, VP2, and RdRp complete genes conducted on the basis of 238, 216, and 215 sequences, respectively, showed a slightly different picture: the topology of phylogenetic trees indicated that representatives of the C2 subvariant did not originate from C1, but evolved from an ancestor common to C and D variants that existed in 2003\u0026ndash;2005. The intermediate stage in the course of this evolution were the isolates MK907790_G19_020_FR_2014 and MW661253_BMH15-067_Ca_2015, which we combined into the C1/2 subvariant.\u003c/p\u003e \u003cp\u003eFive isolates assigned to the C2 subvariant and two \u0026ndash; to the C1/2 subvariant were previously included in the analysis of almost whole nucleotide sequences conducted by Dinu et al. (2023) during the study of a large outbreak of norovirus infection in Romania in 2021 [70]. The authors showed that these genomes fall into a distinct P.17-polymerase-type clade/cluster, different from the previously described Kawasaki clades/clusters, but noted that it is hard to determine if the potential new clades identified in its study represent transitional variants or successful, well-established variants.\u003c/p\u003e \u003cp\u003eThe fact that these seven isolates belong to C variant, and not to distinct new variants, as suggested by Dinu (2023), is, in our opinion, confirmed by the assessment of the percentage of differences in the amino acid sequences of the VP1 protein of these isolates, which is 2.26% with representatives of C variant and significantly more with representatives of D variants (4.42%), A (10.69%) and B (12.48%). A criterion for identifying individual variants within a genotype, accounting for more than 4.1-5% differences in the complete amino acid sequence of the VP1 protein, was proposed by the International Norovirus Classification Group for GII.4 norovirus variants and extrapolated to other genotypes [16, 40, 84].\u003c/p\u003e \u003cp\u003eOur data showed that C2 subvariant has spread in Russia and is also circulating in Europe, the USA and Latin America (Brazil). Noroviruses, the sequences of which cluster with the Nizhny Novgorod strain ON854112_173_NN_RU_2022, judging by the phylogenetic trees presented in the article by Eftekhari M. et al. (2023), were also detected in Iran in 2021 [85].\u003c/p\u003e \u003cp\u003eThe new subvariant has a number of substitutions in the amino acid sequence of the VP1 protein, located near conformational antigenic epitopes blocked by neutralizing antibodies. These substitutions distinguish them from both representatives of the C1 subvariant and from representatives of the D variant, which has been widespread in recent years, and may facilitate evasion of previously formed immunity to GII.17 norovirus.\u003c/p\u003e \u003cp\u003eThe critical importance of five amino acid residues (namely 293Q, 294I, 295N, 296Q and 299R) that form the blocking epitope of cluster IIIb (D variant) was highlighted by Yi et al. (2021) [76]. Liao et al. (2022) also considered the presence of glutamine (Q) at position 298 (296 considering deletions compared to A and B variants) as a key feature of D variant [77]. As shown in our study, C2 subvariant is completely different from D variant in this epitope, having 293E, 294T, 295D and 299K, the same as C1 variant, and 296N, the same as strain Tokyo_JP_1976. In addition, the presence of 297P in all representatives of C2 and C1/2 also brings them closer to strain Tokyo_JP_1976.\u003c/p\u003e \u003cp\u003eThe strains of the C2 and C1/2 subvariants have a HBGA binding site carrying the V444/442Y substitution. The presence of tyrosine (Y) in this position, as shown by Qian et al. (2018), allows new variants of the GII.17 genotype (C and D) to bind effectively to host receptors, unlike representatives of variants A and B [9]. Early strains carrying valine (V) in this position, which does not provide the necessary Van der Waals interaction with fucose, showed weak ability to bind host HBGA. It is interesting to note that of all the early GII.17 norovirus strains, only the Tokyo_JP_1976 strain also has tyrosine in this position.\u003c/p\u003e \u003cp\u003ePreviously, based on phylogenetic analysis of ORF2 gene sequences and amino acid substitutions in the VP1 protein, it was suggested that C variant, which emerged in 2012, could have originated from a virus similar to Tokyo_JP_1976 [86, 87], but for unknown reasons it was not detected for 37 years [9]. The results obtained in our study showed the return to active circulation of C variant, which had not circulated since January 2016. Data on amino acid substitutions in subvariant C2, which bring it even closer to the isolate Tokyo_JP_1976, indicate that the evolution of this phylogenetic lineage has continued in the last decade, has the character of convergence with the ancestral strain, and for four years (2017\u0026ndash;2020) these processes were latent.\u003c/p\u003e \u003cp\u003eAccording to the CDC website, genotype II.17 came in second place after GII.4 in the 2022\u0026ndash;2023 epidemic season [88]. An increase in the share of genotype GII.17 in the Russian Federation was noted in 2022 and 2023 [89]. However, the emergence of this variant did not have a noticeable impact on the incidence of norovirus infection. Our calculation of the effective population size using the example of four genome regions confirmed the results of a similar analysis by Sang et al. (2018), obtained on the basis of the VP1 gene, which indicated the existence of an epidemic peak in 2014\u0026ndash;2015 during the period of the appearance and spread of C and D variants [40]. But, as we have shown, the subsequent appearance of a new C2 subvariant and its circulation in a number of territories in 2021\u0026ndash;2023 did not affect the effective population size.\u003c/p\u003e \u003cp\u003eThe analysis of partial genome sequences of noroviruses, including the overlap zone of non-structural and structural protein genes (RdRp-VP1), conducted in this study also showed the return to circulation in 2018 of the B variant of the GII.17 genotype, which had not been detected worldwide since 2009. Noroviruses with the GII.17/B capsid were detected in wastewater in South Africa in 2018\u0026ndash;2022, in association with P7 and P17 polymerases [66]. The evolution of this variant, most likely in a latent form, continued on the African continent. More extensive nucleotide sequences of the genome of these noroviruses are needed for a detailed analysis.\u003c/p\u003e \u003cp\u003eThe obtained results provide additional information to characterize the evolutionary features of noroviruses of different genotypes. The concept of evolving and static genotypes of noroviruses was previously proposed [39]. Only GII.4 was classified as evolving, since the 1990s it had a change of epidemic variants every 2\u0026ndash;3 years, with the previous variant being completely replaced by the next one, and by 2012 there were already eleven variants of this genotype. Static genotypes have a significantly smaller number of variants, and they circulate for many years, evolving in parallel. In particular, we confirmed this for the GII.6 genotype [90].\u003c/p\u003e \u003cp\u003eHowever, the GII.4 Sidney variant has been circulating in the world for more than 10 years since 2012 and evolves not due to mutations in the gene of the major structural protein, which lead to the formation of new epidemic variants, but due to the acquisition of other genes of non-structural proteins in the process of recombination [91]. New variants of the GII.4 genotype \u0026ndash; Hong Kong2019[P31] and San-Francisco_2017[P31] \u0026ndash; are antigenically different from previously circulating ones, have been identified in a number of countries in Europe and Asia, but have been circulating for more than 7 years without causing huge outbreaks. It is not yet clear, whether the pandemic potential of these variants will be realized or whether they are an example of suppressed circulation of minor norovirus variants [92].\u003c/p\u003e \u003cp\u003eGenotype GII.17, on the contrary, showed the features of an evolving genotype, when C variant was suddenly replaced by the dominant D variant in 2014\u0026ndash;2015 shortly after its emergence in 2012. The latter circulated as a minor in the following years. However, the return to active circulation of C variant in 2022\u0026ndash;2023 shown in our study, as well as the discovery of isolates of B variant after almost a 10 year break, indicates that the co-circulation of several independently evolving variants of the GII.17 genotype continues. This fact, in all probability, confirms its belonging to static genotypes and is consistent with the assumption that non-GII.4 noroviruses are characterized by linear evolution without substitution of variants. This evolution is due to minimal changes at the protein level due to a higher ratio of synonymous substitutions compared to non-synonymous ones, since VP1 of these viruses is under strong evolutionary constraints and cannot accumulate amino acid substitutions [87].\u003c/p\u003e \u003cp\u003eTo better understand the spatiotemporal dynamics of this genotype, as well as to study evolutionary processes in the norovirus population in general, continuous surveillance of their circulation and retrospective studies based on archival samples are necessary.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e NVE conceived and designed the experiments, wrote the manuscript. SVO and AEA performed the experiments. NVE and OVM analyzed the data. TAS and NAN revised the manuscript. All authors revised and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported by the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e This study was approved by the Ethics Committee of the I.N. Blokhina Nizhny Novgorod Research Institute of Epidemiology and Microbiology of the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing. The patient identities were de-linked from their unique laboratory identifiers to ensure confidentiality.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Kapikian AZ, Wyatt RG, Dolin R, Thornhill TS, Kalica AR, Chanock RM (1972) Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. J Virol 10(5):1075\u0026ndash;1081. https://doi.org/10.1128/jvi.10.5.1075-1081.1972\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lopman BA, Steele D, Kirkwood CD, Parashar UD (2016). The Vast and Varied Global Burden of Norovirus: Prospects for Prevention and Control. PLoS Med. 13(4):e1001999. 10.1371/journal.pmed.1001999\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Carlson KB, Dilley A, O'Grady T, Johnson JA, Lopman B, Viscidi E (2024) A narrative review of norovirus epidemiology, biology, and challenges to vaccine development. 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Blokhina Nizhny Novgorod Research Institute of Epidemiology and Microbiology","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Norovirus, Genetic diversity, Genotyping, Phylogenetic analysis, Variant, Subvariant","lastPublishedDoi":"10.21203/rs.3.rs-5393670/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5393670/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNorovirus is one of the leading causes of acute gastroenteritis worldwide and is characterized by significant genetic diversity. In this study, based on phylogenetic analysis of the genome sequences of noroviruses circulating in Nizhny Novgorod in 2014\u0026ndash;2023, as well as those retrieved from the GenBank database, the return to active circulation of the C variant of the GII.17[P17] genotype, displaced in 2015\u0026ndash;2016 by the D variant, is shown. A new subvariant C2, different from the C1 subvariant circulating in the middle of the last decade, was identified. Amino acid substitutions characteristic of C2 were found in the main structural protein VP1, bringing it closer to the Tokyo_JP_1976 strain identified in the 1970s. It was established that the C2 subvariant circulated in 2021\u0026ndash;2023 in European and American countries and caused outbreaks of norovirus infection. The data obtained indicate that the evolution of the phylogenetic lineage represented by the C variant of the GII.17 genotype has been continuing in the last decade and has the character of convergence with the ancestral strain, and for four years (2017\u0026ndash;2020) these processes were latent.\u003c/p\u003e","manuscriptTitle":"Re-emergence and Spread of Norovirus Genotype Gii.17 Variant C in 2021-2023","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-07 05:27:09","doi":"10.21203/rs.3.rs-5393670/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f5e1eb09-0352-429d-8639-f8883407415f","owner":[],"postedDate":"November 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39880375,"name":"Virology"}],"tags":[],"updatedAt":"2024-11-07T05:27:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-07 05:27:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5393670","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5393670","identity":"rs-5393670","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}