Nothing Special   »   [go: up one dir, main page]
Skip to main content
Download PDF

Complete genome characterization by nanopore sequencing of rotaviruses A, B, and C circulating on large-scale pig farms in Russia

Virology Journal volume 21, Article number: 289 (2024) Cite this article

Abstract

Background

Rotaviruses are the major etiological agents of gastroenteritis and diarrheal outbreaks in plenty of mammalian species. The genus Rotavirus is highly diverse and currently comprises nine genetically distinct species, and four of them (A, B, C, and H) are common for humans and pigs. There is a strong necessity to comprehend phylogenetic relationships among rotaviruses from different host species to assess interspecies transmission, specifically between humans and livestock. To reveal the genetic origin of rotaviruses from Russian pig farms, nanopore-based metagenomic sequencing was performed on the PCR-positive specimens.

Methods

Samples were selected among the cases submitted to routine diagnostic or monitoring studies to the Laboratory of Biochemistry and Molecular Biology of “Federal Scientific Center VIEV” (Moscow, Russia). The selected positive samples were genotyped using nanopore sequencing method.

Results

Five porcine RVA isolates were completely sequenced, and genotype analysis revealed various porcine G/P genogroups: G2, G3, G4, G5, G11 and P[6], P[7], P[13], P[23], P[27] with a typical backbone constellation I5-R1-C1-M1-A8-N1-T1/7-E1-H1. The RVB isolate was detected in combination with RVA in a rectal swab from a diseased pig in Krasnoyarsk Krai. It was characterized by the following genogroups: G15-P[X]-I11-R4-C4-M4-A8-N10-T4-E4-H7. The first complete porcine RVC genome from Russia was obtained with genomic constellation G6-P[5]-I14-R1-C1-M1-A7-N9-T6-E1-H1, and the phylogenetic analysis revealed putative novel genotype group for the VP6 gene-I14. Additionally, the first porcine kobuvirus isolate from Russia was phylogenetically characterized.

Conclusions

The applied nanopore sequencing method successfully genotyped the RV isolates and additionally revealed co-circulated species. The study demonstrates high genetic variability of Russian RVA isolates in VP4/VP7 genes and phylogenetically describes local RVB and RVC. Complete characterization of genomic segments is a crucial methodology in tracing the rotavirus's evolution and evaluating interspecies transmissions.

Introduction

Rotaviruses (RVs) are the principal causative agents of gastroenteritis and acute diarrhea in young animals worldwide. The rotavirus genome is enclosed in a triple-layered icosahedral capsid and represented by segmented double-stranded RNA (dsRNA) of eleven segments encoding six structural (VP1-VP4, VP6, and VP7) and five or six nonstructural (NSP1-NSP5/6) proteins [1]. RVs are characterized by two genetic mechanisms: genomic and antigenic drift and shift. Drift consists of point mutations in the genes due to recombination that ultimately lead to antigenic changes. Antigenic shift occurs by reassortment of genomic segments when two or more viral strains co-infect the same cell, producing new gene combinations as a result [2]. Both of these processes constantly take place in nature and significantly drive RV diversification into multiple distinct genotypes, facilitating interspecies transmission.

To date, RVs are members of the Sedoreoviridae family and classified into nine species, A to J, based on the antigenic properties and sequence diversity of the inner viral capsid protein (VP6) [3]. In reference to the genetic and antigenic differences of the outer capsid proteins (VP4 and VP7), which induce neutralizing antibodies, RVs are divided into G and P types. This dual typing system plays a substantial role in the epidemiological studies directed at preventive control measures for RV infections. Furthermore, due to the high genetic variation and reassortment ability of RV strains, this dual (G/P) typing system was extended to a full-genome sequence classification, taking into account all eleven genomic segments. Initially, such classification for the comparison of complete genomes based on established nucleotide percent cut-off values was developed for RVA, where Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx represents the genotypes of the genes VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6, respectively [4]. Subsequently, with the gradual accumulation of genomic data, full-genome classifications were proposed for other RV species [5,6,7].

Four RV species (RVA, RVB, RVC, and RVH) are common for pigs and humans and create a potential risk of zoonotic transmission. RVA is the most prevalent RV species and a major causative agent of diarrhea in pigs, primarily in nursery and postweaning periods. The virus is extremely diverse and inflicts serious losses on farm productivity and swine welfare. At least, twelve G genotypes (G1–G6, G8–G12, and G26) and 16 P genotypes (P[1] to P[8], P[11], P[13], P[19], P[23], P[26], P[27], P[32], and P[34]) of RVA have been associated with infections or diseases in pigs. Notably, porcine G genotypes of G3, G4, G5, G9, and G11 are ordinarily combined with the P genotypes of P[5], P[6], P[7], P[13], P[28], and P[32] [8]. In humans, RVA strains are characterized by two main genotype constellations, Gx-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 and G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, and are called Wa-like and DS-1-like strains, respectively [9]. It appears that human Wa-like and porcine RVA strains possess a common evolutionary ancestor because genotype 1 genes are frequent for strains from both species [4]. As a severe pathogen for humans and pigs, RVA is presently the most studied RV species, and its circulation and genetics are research subjects globally.

Until recently, the pathogenic role of RVB in diarrheal outbreaks has remained unclear because of the concomitant infections with other diarrhea-associated viruses and the difficulties of RVB adaptation in vitro. Nonetheless, for the last decade, diarrheal outbreaks have often been connected with the RVB presence, mainly in suckling piglets [10, 11]. There are currently relatively few complete genomes of RVB strains derived from pigs; hence, the genetic variety of the virus is incompletely defined. The vast majority of deposited sequenced strains originate from the USA and Japan, while the diversity of European strains is merely limited by several isolates from Spain and one from Croatia [12, 13].

RVC was originally identified in pigs with diarrheal syndrome in 1980 [14]. Subsequently, RVC was detected in cases associated with gastroenteritis in a wide range of species, such as cows, ferrets, dogs, and also humans [15,16,17,18]. Porcine RVC has been often identified in cases of diarrhea outbreaks in suckling and weaned piglets [19], but the virus also circulates among post-weaning pigs, possessing pathogenic exposure [20]. During the last few years, the number of complete RVC genomes has dramatically increased, displaying the enormous genetic variability of this species [7].

To the present date, information regarding the complete genome characteristics of porcine RVs from Russia is severely limited. An outbreak of enteric disease among suckling piglets in 2015 led to the determination of one complete porcine RVB genome (Buryat15 strain) [10]. Recently, single porcine RVH has been detected in asymptomatic piglets and genetically characterized [21]. Works introducing the circulation and genetic diversity of RVA and RVC in Russia are dedicated solely to infections in human populations [22,23,24], while porcine are not well researched.

RVs are not a single cause of diarrheal infections in pigs, while other viral and bacterial species can contribute both as primary and secondary pathogens. Among the most significant viral agents involved in enteric infections are other RNA viruses, such as members of the Coronaviridae family, particularly porcine epidemic diarrhea virus (PEDV) and transmissible porcine gastroenteritis virus (TGEV) [25]. Apart from coronaviruses and RVs, viruses of the genera Kobuvirus, Astrovirus, Enterovirus, Sapelovirus, Torovirus, and several others have been detected in porcine fecal virome [12, 26]. Simultaneously, the connection between their presence and the development of diarrhea in pigs has not yet been established. The bacterial enteric pathogens mostly belonged to the following genera: Escherichia, Clostridium, Salmonella, and Brachyspira [27].

In this work, we applied nanopore sequencing to obtain genome sequences of RVA, RVB, and RVC from pigs of different age groups breeding on several Russian pig farms. We classified the isolates in compliance with the full-genome classifications and phylogenetically compared them with openly available nucleotide sequences to elucidate the origin of their genomic segments. Additionally, the spectra of co-circulating species were inspected using a metagenomic approach.

Materials and methods

Collection of samples from pig herds

The study was performed with samples received as part of a routine diagnostic or monitoring protocol during 2022–2023 at the Laboratory of Biochemistry and Molecular Biology of the “Federal Scientific Center VIEV” (Moscow, Russia). The samples were collected from pigs of different ages and healthy statuses from three large-scale industrialized pig farms with a farrow-to-finish production system. The farms were located in both European (Moscow Oblast) and Asian (Krasnoyarsk Krai and the Republic of Buryatia) parts of Russia. The transportation of specimens to the laboratory was performed under freezing conditions. Six RV-positive samples with a low Ct value in qPCR (< 20) were chosen for nanopore-based metagenomic sequencing in order to genotype RV species and investigate additional spectra of viral and bacterial species (Table 1).

Table 1 Characteristics of porcine samples selected for the nanopore-based metagenomic sequencing
Full size table

Nanopore sequencing and genome assembly

Nanopore metagenomic sequencing was conducted in accordance with the protocol developed by PathoSense BV (Merelbeke, Belgium), as was described previously [21]. Briefly, it included nucleic acid extraction using the Quick-DNA/RNA viral kit (Zymo Research, CA, USA), reverse transcription using the SuperScript IV Reverse Transcriptase (ThermoFisher Scientific, USA) with following enrichment by PCR (KAPA HiFi HotStart ReadyMix; Roche, Switzerland). Then amplicons were cleaned with the magnetic beads (AMPure XP; Beckman Coulter, USA) in a ratio 1:1. Final quantity and quality were verified using NanoDrop OneC spectrophotometer (ThermoFisher Scientific, USA). The preparation of the sequencing libraries was carried out using the Rapid Sequencing and Rapid Barcoding kits (ONT, UK). Nanopore sequencing was performed on a MinION platform using a flow cell R9.4.1 (ONT, UK) for 6 h.

Fast5 files were obtained using MinKNOW software (v.23.11.2) and then basecalled and demultiplexed using Guppy basecaller (v.6.0.1 + 652ffd1) in the supper accurate mode settings (-c dna_r9.4.1_450bps_sup.cfg). Subsequently, raw sequencing files were quality filtered (Q-score ≥ 7) using NanoFilt v2.8.0, and host swine reads were removed using minimap2 (v2.24) [28] and samtools (v1.19.2) [29]. Filtered reads were taxonomically classified using BLASTx and BLASTn algorithms according to the customized viral and bacterial databases, and the hits with the lowest e-values were visualized with KronaTools v2.7 [30]. For genome assembly of RVs, obtained filtered reads were binned according to their gene segments using minimap2 (v2.24) and samtools (v1.19.2). De novo genome assembly was performed individually for each gene segment using canu (v2.2) [31] and medaka polishing (v1.6.1; ONT). The minimum coverage parameter for the assembly was 30 × . To evaluate genome assemblies, consensus sequences were created using samtools (v1.6) and iVar (v1.4.2) [32]. Additionally, sequences were checked manually using IGV (v2.13.2) software [33].

Genotyping and phylogenetic analysis

Sequence alignments were created using the ClustalW algorithm. Phylogenetic analysis was conducted using the maximum-likelihood method with the general time reversible (GTR) nucleotide substitution model supported by 1000 bootstrap replicates using the MEGA software (v7.0) [34]. Subsequently, phylogenetic dendrograms were exported as Newick files and then annotated in iTOL v6 [35]. For the comparable analysis, RV isolates were selected using BLASTn search in the NCBI GenBank database. Genetic distances were calculated using the Kimura two-parameter correction at the nucleotide level. The isolates were genotyped in compliance with the previously proposed full-genome classification systems with specified nucleotide percent cut-off values [4, 5, 7].

Results

During the monitoring studies at the Laboratory of Biochemistry and Molecular Biology of “Federal Scientific Center VIEV”, six RV-positive samples from both healthy and diseased pigs of different age groups were selected for complete genotyping using nanopore sequencing. As a result, we characterized segments of the detected porcine RVs from the positive specimens, and the obtained sequences were deposited in the NCBI GenBank. Phylogenetic dendrograms were inferred for each gene segment from assembled sequences in this study along with reference sequences in the GenBank. The genomic constellations and accession numbers of sequenced RVs are represented in Table 2. Additional spectra of the most abundant viral and bacterial species are represented in the Supplementary File 1.

Table 2 Genome constellation and GenBank accession number for each RV strain
Full size table

Genetic diversity and genotyping of RVA isolates

Five porcine RVA isolates were completely sequenced and classified in accordance with the criteria proposed in previous studies [4]. A wide variety of G/P genotypes was detected for the VP7 and VP4 encoding genes, and the following genome constellation was determined for the backbone genes: I5-R1-C1-M1-A8-N1-T7-E1-H1. According to the phylogenetic analysis, Russian RVA isolates were assigned to five different G-groups: G2, G3, G4, G5, and G11 for the outer capsid glycoprotein VP7, and among P-groups, the following genotypes were detected: P[6], P[7], P[13], P[23], and P[27] (Fig. 1).

Fig. 1
figure 1

Phylogenetic dendrograms constructed with the GTR model for the VP4 and VP7 RVA genes. The RVA isolates identified in this study are colored in bold blue. Scale bars indicate nucleotide substitutions per site. Bootstrap values greater than 80% are specified. GenBank accession number, host, country, and collection year of each isolate are also shown. RVA genotypes are specified on the right of the bracket

Full size image

The genes VP1, VP2, VP3, NSP2, NSP4, and NSP5 in all sequenced RVA isolates were assigned to genotype 1 genes, designating the Wa-like rotavirus genogroup, but phylogenetically, they predominantly constituted peculiar clusters with other porcine isolates that were distinct from the human ones (Supplementary Fig. 1). Despite the fact that the NSP3 gene was represented by the T7 genotype in all of the Russian isolates, it is also noteworthy that the T1 genotype was additionally encountered in the sample from Krasnoyarsk Krai. The RVA isolates from the Republic of Buryatia were originated from the same farm but from different pig farrowing barns. These isolates were characterized by high nucleotide identity in gene segment sequences (99.7–100%), excluding NSP1, and VP4 and VP7 genes, where the following G/P genotype combinations were determined: G5P[13] and G4P[6]. The mixed RVA infection was observed in a piglet from Krasnoyarsk Krai, as we sequenced two VP7 (G3/G11), three VP4 (P[6]/P[13]/P[23]), and two NSP3 (T1/T7) gene sequences. Combinations G2P[27] and G3P[7] were found in rectal swabs of clinically healthy piglets from the Moscow Oblast. These isolates were detected on the same farm but a year apart and were cognate to each other in VP2, VP3, VP6, NSP4, and NSP5 backbone gene sequences (96.8–98.9%), forming the monophyletic clades (Supplementary Fig. 1).

Phylogenetic analysis of RVB isolate

The RVB isolate was identified in the co-infection with RVA in a rectal swab of a diarrheal weaned piglet from Krasnoyarsk Krai. Complete coding sequences were achieved for all the genomic segments, except VP4, where a partial sequence 1708 bp in length was obtained. The isolate was characterized by the following genotype constellation: G15-P[X]-I11-R4-C4-M4-A8-N10-T4-E4-H7. According to the BLASTn search and phylogenetic analysis, the VP4 gene was relatively close to sequence of the Spanish isolate (GenBank MK953165) that was described with an unclassified P[X] genotype [12]. The VP7 gene was related to G15 genotype, which was previously detected only in the single Japanese isolate (GenBank AB490435), and the VP6 gene was clustered with isolates from the USA and classified as infrequent I11 genotype (Fig. 2). The backbone gene composition was consistent with the majority of previously genotyped North-American isolates. In the NSP3, NSP4, and NSP5 genes, the isolate was placed within the common clade with the Buryat15 strain, which was detected in the Buryatia Republic of Russia in 2015 (Supplementary Fig. 2).

Fig. 2
figure 2

Phylogenetic dendrograms constructed with the GTR model for the VP4, VP6, and VP7 RVB genes. The RVB isolate identified in this study is colored in bold blue. Scale bars indicate nucleotide substitutions per site. Bootstrap values greater than 80% are specified.. GenBank accession number, host, country, and collection year of each isolate are also shown. RVB genotypes are specified on the right of the bracket

Full size image

Phylogenetic analysis of RVC isolate

RVC was the only viral species detected in the rectal swab of the seven-day-old suckling piglet with diarrheal syndrome. Taking into account the updated system with specified cut-off values and constructed phylogenetic dendrograms, we classified the detected RVC isolate as follows: G6-P[5]-I14-R1-C1-M1-A7-N9-T6-E1-H1 (Fig. 3). The phylogenetic analysis revealed that the genomic segments belonged to the porcine-origin clusters but geographic dependence between the isolates was not observed (Supplementary Fig. 3).

Fig. 3
figure 3

Phylogenetic dendrograms constructed with the GTR model for the VP4, VP6, and VP7 RVC genes. The RVC isolate identified in this study is colored in bold blue. The putative novel VP6 genotype I14 is additionally highlighted in light green. Scale bars indicate nucleotide substitutions per site. Bootstrap values greater than 80% are specified. GenBank accession number, host, country, and collection year of each isolate are also shown. RVC genotypes are specified on the right of the bracket

Full size image

According to the proposed cut-off value for the VP6 gene (86%), we calculated the genetic distances for the segments and separated a novel potential genotype group I14 (Supplementary File 2). The finding was additionally confirmed with excellent bootstrap support (99%). The comparison with other G6P[5] RVC isolates revealed high genetic flexibility in the backbone genes (Table 3).

Table 3 Genotype constellation of several characterized porcine G6P[5] RVC isolates, including the studied one. The RVC isolate identified in this study is indicated in bold
Full size table

Genetic characterization of porcine kobuvirus

Porcine kobuvirus (PKV) was detected in a virome of clinically healthy piglets from Moscow Oblast. The partial nucleotide sequence equaled 2271 nt in length of the start region of the polyprotein gene was assembled and phylogenetically compared with publicly available nucleotide sequences (Fig. 4). The BLASTn analysis of the virus sequence demonstrated that the closest isolates shared nucleotide similarity in a range of 88.29–89.05% and originated from Spain (isolate 3330; GenBank: KT892968) and China (isolate Y-1-CHI; GenBank: GU292559).

Fig. 4
figure 4

Phylogenetic dendrogram constructed with the GTR model for the partial sequence (2271 nt) of Porcine kobuvirus (PKV). The PKV isolate identified in this study is colored in bold blue. Bootstrap values greater than 80% are specified. GenBank accession number, country and collection year of each isolate are shown in brackets on the right side

Full size image

Discussion

RVs have been recognized as an important cause of diarrhea in suckling and weaned piglets, but they can circulate both in animals with clinical manifestations and inadvertently in subclinical cases [36]. Currently, there is a lack in knowledge regarding the genetic diversity of RVs in pig herds in Russia. In order to increase this knowledge, we performed whole-genome sequence analysis of several porcine RVs circulating on farrow-to-finish pig farms in the country.

Five porcine RVA isolates from three distinct Russian regions were completely sequenced and genotyped. The following G/P combinations were identified in the samples of deceased and diseased piglets with diarrheal syndrome: G5P[13] and G4P[6] subtypes from the Republic of Buryatia, evidencing the reassortment event and a set of co-circulating G3/G11/P[6]/P[13]/P[23] subtypes from Krasnoyarsk Krai. According to some monitoring studies, these G/P groups are commonly spread in pig herds and often coupled with diarrhea in various age groups [37,38,39]. By contrast, combinations G2P[27] and G3P[7] were detected in rectal swabs of clinically healthy piglets from the Moscow Oblast. Subtype G2P[27] is not very frequent in swine herds and was found in several European countries, Thailand, and Canada, not only in asymptomatic infections in pigs but also with diarrhea [27, 40,41,42]. However, the G3P[7] subtype was previously detected in the samples obtained predominantly from diarrheal pigs in the USA, Spain, Belgium, Croatia, and Chile [38, 43,44,45]. The genes VP1, VP2, VP3, NSP2, NSP4, and NSP5 were classified into the following genotypes: R1, C1, M1, N1, E1, and H1, respectively, showing the stable genotype composition correlating with the Wa-like human genogroup. Nevertheless, the porcine and human Wa-like lineages apparently form individual evolutionary routes that are clearly being viewed in the constructed phylogenetic dendrograms. On the other hand, the existence of the I5, A8, and T7 genotypes for VP6, NSP1, and NSP3 among the RVA isolates indicates distinctive genetic drift. Currently, I5 and A8 genotypes are widely dispersed in herds and are thought to be typical porcine genotypes [43, 46,47,48]. Regarding the NSP3 gene, the T7 genotype has become widespread among pigs for the last years and gradually supersedes the T1 [49,50,51]. According to our study, the T7 genotype has firmly consolidated in Russian pig herds, as evidenced by its considerable dominance among genotyped isolates. Such a global phenomenon can be presumably explained by the fact that, in contrast to Wa-like genes, this particular gene cluster promotes better viral replication in cells, ensuring rotavirus survivability [47]. Thus, the obtained data as a whole reflects the genetic heterogeneity of Russian RVA isolates in VP4 and VP7 due to reassortment events, while demonstrating a conservative picture in the structure of the backbone genes that is consistent with numerous works from other world localities [46, 47, 50].

RVB isolate was detected in co-infection with several RVA subtypes; therefore, its pathogenic role in this case was not obvious. The isolate was characterized by extensive diversity in VP4, VP7, and VP6 genes while demonstrating typical porcine-like backbone gene composition R4-C4-M4-A8-N10-T4-E4-H7. As was determined earlier, the majority of isolates from the USA shared the identical internal core gene cassette, forming North-American genogroups, in contrast to Asian ones, where some of the isolates, which originated from India, Japan, and Vietnam, built up additional phylogenetic clusters in several non-structural protein genes [5]. This conserved genotype pattern was similarly seen in the previously identified Russian strain, Buryat15, and recently characterized isolate from South Africa [10, 50]. The European isolates from Spain and Croatia possessed a comparable combination, but with some variations in the VP1 and VP2 genes, which were newly related to the R6/7 and C6 genogroups, respectively [13]. However, the porcine RVB strain HNLY-2022 from China demonstrated that more than half of its genome segments were related to previously unknown genotypes [11]. These results suggest that more research is necessary because the current knowledge is insufficient to completely assess the genetic diversity of RVBs.

RVC is another causative agent associated with enteric problems in neonatal pigs. In our study, the RVC genome was completely sequenced from a sample of a suckling piglet with clinical signs of enteric disease. The RVC was the sole viral species in the virome and was suspected as a causative agent of diarrhea in that case. The detected G6P[5] subtype is quite abundant in diarrheal pigs [12, 52, 53]. In general, porcine RVC isolates, in contrast to RVA and RVB, are characterized by more high genetic heterogeneity not only in VP4/VP7 but in the great majority of other genes, with the exceptions being VP1 and NSP5 [7]. The identified RVC isolate was as well characterized by various genotype groups in the backbone constellation in comparison with other G6P[5] isolates (Table 3). There is an opinion that pigs serve as a reservoir host of RVC [54], and such diversification indicates a high level of reassortment potential in this species. RVC full-genome classification was originally proposed by Suzuki and Hasebe [6] and then updated by Wang et al. [7]. In this study, we suggested a putative novel I14 genotype for the VP6 gene. However, it is important to note that more sequencing data are required before this distinct split can be considered conclusively established.

PKV, within the Picornaviridae family, is considered a causative agent of diarrheal outbreaks in pigs, but its pathogenicity is still ambiguous as the virus has been detected in both healthy and diseased animals. Moreover, the co-infection of PKV with other viruses, including RVs, has been found frequently, which complicates the virus's possible pathogenic role in diarrhea [55, 56]. During the metagenomic analysis, we identified the virus in a sample of a clinically healthy suckling piglet. To the best of our knowledge, this is the first PKV sequence from Russia, but more in-depth epidemiological analyses need to be performed in order to evaluate the virus prevalence in the local farms.

In summary, this work delineates the first genome-wide research, targeting porcine RV diversity on Russian large-scale farms. The study revealed high genetic variability in VP4/VP7 genes among RVA isolates circulating in pig herds. In the case of RVB and RVC, our work increased the current insight of the existing local gene compositions. According to the phylogenetic analysis, all the RV isolates were clustered into porcine genogroups, evidencing the absence of cross-species reassortment events. It’s hard to draw a precise parallel between RV subtype and pathogenicity, but it has to be mentioned that clinically healthy pigs can act as reservoirs for generating novel viral combinations, which entails a risk of further pathogen transmission. With recent advances in sequencing techniques, this data emphasizes that the integration of such cutting-edge methods in monitoring studies on a regular basis could comprehensively promote our understanding of reassortment capability and cross-species transmissions of RVs.

Availability of data and materials

Viral nucleotide sequences are available at the NCBI GenBank under the accession numbers PP874392-PP874402, PP874404-PP874462, PP898323-PP898333, and PP935171.

References

  1. Desselberger U. Rotaviruses. Virus Res. 2014;190:75–96.

    Article  CAS  PubMed  Google Scholar 

  2. McDonald SM, Nelson MI, Turner PE, Patton JT. Reassortment in segmented RNA viruses: mechanisms and outcomes. Nat Rev Microbiol. 2016;14:448–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Matthijnssens J, Attoui H, Bányai K, Brussaard CP, Danthi P, Del Vas M, et al. ICTV virus taxonomy profile Sedoreoviridae 2022. J Gen Virol. 2022;103(10):001782.

    Article  CAS  Google Scholar 

  4. Matthijnssens J, Ciarlet M, Heiman E, Arijs I, Delbeke T, McDonald SM, et al. Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains. J Virol. 2008;82:3204–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Shepherd FK, Herrera-Ibata DM, Porter E, Homwong N, Hesse R, Bai J, et al. Whole genome classification and phylogenetic analyses of rotavirus B strains from the United States. Pathogens. 2018;7:44.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Suzuki T, Hasebe A. A provisional complete genome-based genotyping system for rotavirus species C from terrestrial mammals. J Gen Virol. 2017;98:2647–62.

    Article  CAS  PubMed  Google Scholar 

  7. Wang Y, Porter EP, Lu N, Zhu C, Noll LW, Hamill V, Brown SJ, Palinski RM, Bai J. Whole-genome classification of rotavirus C and genetic diversity of porcine strains in the USA. J Gen Virol. 2021;102(5):001598.

    Article  CAS  Google Scholar 

  8. Vlasova AN, Amimo JO, Saif LJ. Porcine rotaviruses: epidemiology, immune responses and control strategies. Viruses. 2017;9:48.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Matthijnssens J, Van Ranst M. Genotype constellation and evolution of group A rotaviruses infecting humans. Curr Opin Virol. 2012;2:426–33.

    Article  CAS  PubMed  Google Scholar 

  10. Alekseev KP, Penin AA, Mukhin AN, Khametova KM, Grebennikova TV, Yuzhakov AG, et al. Genome characterization of a pathogenic porcine rotavirus B Strain identified in Buryat Republic, Russia in 2015. Pathogens. 2018;7:46.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Li Q, Wang Z, Jiang J, He B, He S, Tu C, et al. Outbreak of piglet diarrhea associated with a new reassortant porcine rotavirus B. Vet Microbiol. 2024;288:109947.

    Article  CAS  PubMed  Google Scholar 

  12. Cortey M, Díaz I, Vidal A, Martín-Valls G, Franzo G, Gómez de Nova PJ, et al. High levels of unreported intraspecific diversity among RNA viruses in faeces of neonatal piglets with diarrhoea. BMC Vet Res. 2019;15:441.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Brnić D, Vlahović D, Gudan Kurilj A, Maltar-Strmečki N, Lojkić I, Kunić V, et al. The impact and complete genome characterisation of viruses involved in outbreaks of gastroenteritis in a farrow-to-finish holding. Sci Rep. 2023;13:18780.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Saif LJ, Bohl EH, Theil KW, Cross RF, House JA. Rotavirus-like, calicivirus-like, and 23-nm virus-like particles associated with diarrhea in young pigs. J Clin Microbiol. 1980;12:105–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mawatari T, Taneichi A, Kawagoe T, Hosokawa M, Togashi K, Tsunemitsu H. Detection of a bovine group C rotavirus from adult cows with diarrhea and reduced milk production. J Vet Med Sci. 2004;66:887–90.

    Article  CAS  PubMed  Google Scholar 

  16. Marton S, Mihalov-Kovács E, Dóró R, Csata T, Fehér E, Oldal M, et al. Canine rotavirus C strain detected in Hungary shows marked genotype diversity. J Gen Virol. 2015;96:3059–71.

    Article  CAS  PubMed  Google Scholar 

  17. Bridger JC, Pedley S, McCrae MA. Group C rotaviruses in humans. J Clin Microbiol. 1986;23:760–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Torres-Medina A. Isolation of an atypical rotavirus causing diarrhea in neonatal ferrets. Lab Anim Sci. 1987;37:167–71.

    CAS  PubMed  Google Scholar 

  19. Amimo JO, Vlasova AN, Saif LJ. Prevalence and genetic heterogeneity of porcine group C rotaviruses in nursing and weaned piglets in Ohio, USA and identification of a potential new VP4 genotype. Vet Microbiol. 2013;164:27–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Roczo-Farkas S, Dunlop RH, Donato CM, Kirkwood CD, McOrist S. Rotavirus group C infections in neonatal and grower pigs in Australia. Vet Rec. 2021;188:e296.

    Article  PubMed  Google Scholar 

  21. Krasnikov N, Yuzhakov A. Interspecies recombination in NSP3 gene in the first porcine rotavirus H in Russia identified using nanopore-based metagenomic sequencing. Front Vet Sci. 2023;10:1302531.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Morozova OV, Sashina TA, Epifanova NV, Kashnikov AY, Novikova NA. Increasing detection of rotavirus G2P[4] strains in Nizhny Novgorod, Russia, between 2016 and 2019. Arch Virol. 2021;166:115–24.

    Article  CAS  PubMed  Google Scholar 

  23. Yuzhakov A, Yuzhakova K, Kulikova N, Kisteneva L, Cherepushkin S, Smetanina S, et al. Prevalence and genetic diversity of group A rotavirus genotypes in Moscow (2019–2020). Pathogens. 2021;10:674.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhirakovskaia E, Tikunov A, Klemesheva V, Loginovskikh N, Netesov S, Tikunova N. First genetic characterization of rotavirus C in Russia. Infect Genet Evol. 2016;39:1–8.

    Article  CAS  PubMed  Google Scholar 

  25. Turlewicz-Podbielska H, Pomorska-Mól M. Porcine coronaviruses: overview of the state of the art. Virol Sin. 2021;36:833–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Puente H, Arguello H, Cortey M, Gómez-García M, Mencía-Ares O, Pérez-Perez L, et al. Detection and genetic characterization of enteric viruses in diarrhoea outbreaks from swine farms in Spain. Porcine Health Manag. 2023;9:29.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Theuns S, Desmarets LMB, Heylen E, Zeller M, Dedeurwaerder A, Roukaerts IDM, et al. Porcine group A rotaviruses with heterogeneous VP7 and VP4 genotype combinations can be found together with enteric bacteria on Belgian swine farms. Vet Microbiol. 2014;172:23–34.

    Article  CAS  PubMed  Google Scholar 

  28. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094–100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. Twelve years of SAMtools and BCFtools. GigaScience. 2021;10:giab008.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Ondov BD, Bergman NH, Phillippy AM. Interactive metagenomic visualization in a Web browser. BMC Bioinf. 2011;12:385.

    Article  Google Scholar 

  31. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27:722–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Grubaugh ND, Gangavarapu K, Quick J, Matteson NL, De Jesus JG, Main BJ, et al. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biol. 2019;20:8.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49:W293–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Smoľak D, Šalamúnová S, Jacková A, Haršányová M, Budiš J, Szemes T, et al. Analysis of RNA virome in rectal swabs of healthy and diarrheic pigs of different age. Comp Immunol Microbiol Infect Dis. 2022;90–91:101892.

    Article  PubMed  Google Scholar 

  37. Lorenzetti E, da Silva Medeiros TN, Alfieri AF, Alfieri AA. Genetic heterogeneity of wild-type G4P [6] porcine rotavirus strains detected in a diarrhea outbreak in a regularly vaccinated pig herd. Vet Microbiol. 2011;154:191–6.

    Article  PubMed  Google Scholar 

  38. Monteagudo LV, Benito AA, Lázaro-Gaspar S, Arnal JL, Martin-Jurado D, Menjon R, et al. Occurrence of rotavirus A genotypes and other enteric pathogens in diarrheic suckling piglets from spanish swine farms. Animals. 2022;12:251.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Chandler-Bostock R, Hancox LR, Nawaz S, Watts O, Iturriza-Gomara M, Mellits KH. Genetic diversity of porcine group A rotavirus strains in the UK. Vet Microbiol. 2014;173:27–37.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Khamrin P, Maneekarn N, Peerakome S, Chan-it W, Yagyu F, Okitsu S, et al. Novel porcine rotavirus of genotype P[27] shares new phylogenetic lineage with G2 porcine rotavirus strain. Virology. 2007;361:243–52.

    Article  CAS  PubMed  Google Scholar 

  41. Steyer A, Poljsak-Prijatelj M, Barlic-Maganja D, Jamnikar U, Mijovski JZ, Marin J. Molecular characterization of a new porcine rotavirus P genotype found in an asymptomatic pig in Slovenia. Virology. 2007;359:275–82.

    Article  CAS  PubMed  Google Scholar 

  42. Lachapelle V, Sohal JS, Lambert M-C, Brassard J, Fravalo P, Letellier A, et al. Genetic diversity of group A rotavirus in swine in Canada. Arch Virol. 2014;159:1771–9.

    Article  CAS  PubMed  Google Scholar 

  43. Neira V, Melgarejo C, Urzúa-Encina C, Berrios F, Valdes V, Mor S, et al. Identification and characterization of porcine Rotavirus A in Chilean swine population. Front Vet Sci. 2023;10:1240346.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Theuns S, Vyt P, Desmarets LMB, Roukaerts IDM, Heylen E, Zeller M, et al. Presence and characterization of pig group A and C rotaviruses in feces of Belgian diarrheic suckling piglets. Virus Res. 2016;213:172–83.

    Article  CAS  PubMed  Google Scholar 

  45. Brnić D, Čolić D, Kunić V, Maltar-Strmečki N, Krešić N, Konjević D, et al. Rotavirus A in domestic pigs and wild boars: high genetic diversity and interspecies transmission. Viruses. 2022;14:2028.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Boene SS, João ED, Strydom A, Munlela B, Chissaque A, Bauhofer AFL, et al. Prevalence and genome characterization of porcine rotavirus A in southern Mozambique. Infect Genet Evol. 2021;87:104637.

    Article  CAS  PubMed  Google Scholar 

  47. Theuns S, Heylen E, Zeller M, Roukaerts IDM, Desmarets LMB, Van Ranst M, et al. Complete genome characterization of recent and ancient Belgian pig group A rotaviruses and assessment of their evolutionary relationship with human rotaviruses. J Virol. 2015;89:1043–57.

    Article  PubMed  Google Scholar 

  48. Silva FDF, Espinoza LRL, Tonietti PO, Barbosa BRP, Gregori F. Whole-genomic analysis of 12 porcine group A rotaviruses isolated from symptomatic piglets in Brazil during the years of 2012–2013. Infect Genet Evol. 2015;32:239–54.

    Article  PubMed  Google Scholar 

  49. Martel-Paradis O, Laurin M-A, Martella V, Sohal JS, L’Homme Y. Full-length genome analysis of G2, G9 and G11 porcine group A rotaviruses. Vet Microbiol. 2013;162:94–102.

    Article  CAS  PubMed  Google Scholar 

  50. Strydom A, Segone N, Coertze R, Barron N, Strydom M, O’Neill HG. Phylogenetic analyses of rotavirus A, B and C detected on a porcine farm in South Africa. Viruses. 2024;16:934.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Vidal A, Clilverd H, Cortey M, Martín-Valls GE, Franzo G, Darwich L, et al. Full-genome characterization by deep sequencing of rotavirus A isolates from outbreaks of neonatal diarrhoea in pigs in Spain. Vet Microbiol. 2018;227:12–9.

    Article  CAS  PubMed  Google Scholar 

  52. Tuanthap S, Phupolphan C, Luengyosluechakul S, Duang-in A, Theamboonlers A, Wattanaphansak S, et al. Porcine rotavirus C in pigs with gastroenteritis on Thai swine farms, 2011–2016. PeerJ. 2018;6:e4724.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Possatti F, Lorenzetti E, Alfieri AF, Alfieri AA. Genetic heterogeneity of the VP6 gene and predominance of G6P[5] genotypes of Brazilian porcine rotavirus C field strains. Arch Virol. 2016;161:1061–7.

    Article  CAS  PubMed  Google Scholar 

  54. Trovão NS, Shepherd FK, Herzberg K, Jarvis MC, Lam HC, Rovira A, et al. Evolution of rotavirus C in humans and several domestic animal species. Zoonoses Public Health. 2019;66:546–57.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Jackova A, Sliz I, Mandelik R, Salamunova S, Novotny J, Kolesarova M, et al. Porcine kobuvirus 1 in healthy and diarrheic pigs: Genetic detection and characterization of virus and co-infection with rotavirus A. Infect Genet Evol. 2017;49:73–7.

    Article  CAS  PubMed  Google Scholar 

  56. Zhou W, Ullman K, Chowdry V, Reining M, Benyeda Z, Baule C, et al. Molecular investigations on the prevalence and viral load of enteric viruses in pigs from five European countries. Vet Microbiol. 2016;182:75–81.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We express sincere gratitude to the participating veterinarians that kindly provided the field samples for this work.

Funding

The study was carried out within the framework of State Assignment No. FGUG-2022–0018 of the Federal State Budget Scientific Institution “Federal Scientific Center VIEV”, Moscow, Russia.

Author information

Authors and Affiliations

  1. Federal State Budget Scientific Institution “Federal Scientific Center VIEV” (FSC VIEV), Moscow, Russia

    Nikita Krasnikov, Alexey Gulyukin, Taras Aliper & Anton Yuzhakov

Authors
  1. Nikita Krasnikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexey Gulyukin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Taras Aliper
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anton Yuzhakov
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.K. performed the sequencing, conducted the bioinformatics and phylogenetic analyses, prepared the figures and tables, and wrote the original draft of the manuscript. A.G. acquired funding. T.A. arranged the sampling process on farms. A.Y. participated in formulation and design of the study, and supervised its execution. All authors were involved in editing the manuscript, reviewed and approved the final version of the manuscript.

Corresponding author

Correspondence to Nikita Krasnikov.

Ethics declarations

Ethical approval and consent to participate

The animal study protocol was approved by the local Ethical and Animal Welfare Committee of the Federal State Budget Scientific Institution “Federal Scientific Center VIEV”, (Moscow, Russia) (Approval number 92/22 from 9 February 2021) for studies involving animals.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Krasnikov, N., Gulyukin, A., Aliper, T. et al. Complete genome characterization by nanopore sequencing of rotaviruses A, B, and C circulating on large-scale pig farms in Russia. Virol J 21, 289 (2024). https://doi.org/10.1186/s12985-024-02567-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12985-024-02567-9

Keywords

Virology Journal

ISSN: 1743-422X