Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.


Introduction
Group A rotavirus (RVA), a member of the Reoviridae family, is the primary pathogen causing acute gastroenteritis in the young of humans and many animal species worldwide. In humans, RVA infections are associated with high morbidity and mortality, being responsible for an estimated 453,000 deaths among children under 5 years of age annually [1]. More than half of these deaths are estimated to occur in developing countries in Asia and Africa [1][2][3]. The mature RVA particle is a triple-layered, non-enveloped icosahedron enclosing an 11-segment genome of double-stranded (ds)RNA [4]. The segmented nature of the genome enables reassortment between/within human and animal strains, and reassortment plays one of the major roles in the generation of the genomic diversity of this medically important virus [5].
Whole genome-based analysis is a reliable method for obtaining precise information on the origin of a given RVA strain, and for tracing its evolutionary pattern [10,17]. To date, the full genome sequences of several DS-1-like G1P [8] strains from Japan and Thailand have been determined and characterized, which indicated the occurrence of reassortment event(s) between Wa-like G1P [8] and DS-1-like G2P [4] human RVAs [13,15,16]. Furthermore, DS-1-like G1P [8] strains in Japan and Thailand are very closely related to one another in all the 11 genes, showing the derivation of these strains from a common origin [16]. However, as the exact evolutionary pattern of DS-1-like G1P [8] strains remains to be elucidated, whole genomic analysis of the DS-1-like intergenogroup reassortant strains having G3P [8] and G2P [8] genotypes, strains SKT-281, SKT-289, and LS-04, might be useful for obtaining a more precise understanding of the evolutionary pattern of DS-1-like G1P [8] strains and related reassortant ones. In this study, deep sequencing using the next generation sequencing (NGS) Illumina MiSeq platform was performed to determine the complete nucleotide sequences of the whole genomes of these three DS-1-like intergenogroup reassortant strains. Furthermore, the whole genomes of six locally circulating human RVAs (two Wa-like G1P [8] and four DS-1-like G2P [4] strains) were also sequenced as references.

Ethics statement
The study was approved by the Ethical Review Committee for Research on Human Subjects of the Ministry of Public Health, Thailand (Ref. no. 10/2555). In this study, written informed consent for the testing of stool samples for RVAs and characterization of identified RVA strains was obtained from the children's parents/guardians.

Virus strains
The full-genomic sequences were determined for strains SKT-281, SKT-289, and LS-04, and locally circulating strains PCB-118, SKT-98, BD-20, NP-M51, SKT-138, and SSKT-133, which were identified in nine stool samples from hospitalized children aged 16-51 months with severe diarrhea in Phechaboon and Sukhothai Provinces, Thailand, during the RVA surveillance program in those provinces in 2012-2014, which involved a total of 3002 fecal specimens (Tacharoenmuang et al., in preparation). Of the 3002 fecal samples, RVA infection was detected in 687 (22.9%). Stool specimens were collected during hospital-based surveillance activities during the RVA vaccine effectiveness evaluation study in Thailand. The stool samples were examined by PCR-based G and P genotyping, and PAGE analysis at the National Institute of Health, Thailand. For Illumina MiSeq sequencing, the samples were submitted to Fujita Health University, Japan. Fecal specimens containing strains SKT-281, SKT-289, LS-04, PCB-118, SKT-98, BD-20, NP-M51, SKT-138, and SSKT-133 were kept at −30°C until use. mA at room temperature, followed by silver staining [20] to determine the genomic dsRNA profiles. For whole genomic analysis, viral dsRNAs were subjected to Illumina MiSeq sequencing as described below.

cDNA library building and Illumina MiSeq sequencing
Preparation of a cDNA library and Illumina MiSeq sequencing were carried out as described previously [3,18,19]. Briefly, a 200 bp fragment library ligated with bar-coded adapters was constructed for strains SKT-281, SKT-289, LS-04, PCB-118, SKT-98, BD-20, NP-M51, SKT-138, and SSKT-133 using an NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (New England Biolabs) and an NEBNext Multiplex Oligos for Illumina (Index Primers Sets 1 and 2) (New England Biolabs) according to the manufacturer's instructions. Library purification was performed using Agencourt AMPure XP magnetic beads (Beckman Coulter). The quality of the purified cDNA library was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Nucleotide sequencing was carried out on an Illumina MiSeq sequencer (Illumina) using a MiSeq Reagent Kit v2 (Illumina) to generate 151 paired-end reads. Data analysis was carried out using CLC Genomics Workbench v8.0.1 (CLC Bio). Contigs were assembled from the obtained sequence reads by de novo assembly. Using the assembled contigs as query sequences, the Basic Local Alignment Search Tool (BLAST) non-redundant nucleotide database was searched to obtain the full-length nucleotide sequence of each gene segment of strains SKT-281, SKT-289, LS-04, PCB-118, SKT-98, BD-20, NP-M51, SKT-138, and SSKT-133. The nucleotide sequences were translated into amino acid sequences using GENETYX v11 (GENETYX).

Phylogenetic analyses
Sequence comparisons were carried out as described previously [3,18,19]. Briefly, multiple alignment of each viral gene was carried out using CLUSTAL W [22]. Phylogenetic trees were constructed using the maximum likelihood method and the Tamura-Nei substitution model using MEGA6.06 [23]. The reliability of the branching order was estimated from 1000 bootstrap replicates [24]. The results of phylogenetic analyses were validated using several other genetic distance models, such as the Kimura 2-parameter, Tamura 3-parameter, Jukes-Cantor, and Hasegawa-Kishino-Yano ones (data not shown).  Nucleotide sequencing and whole-genome-based genotyping of strains SKT-281, SKT-289, and LS-04

Phylogenetic analyses
We next constructed phylogenetic trees using the full-genome sequence for each of the 11 gene segments because phylogenetic analysis of RVA nucleotide sequences provides direct evidence of their relatedness to those of other strains, even within the same genotype [3,5,10] (Figs 3-16). In this study, we employed strains HC12016 [15], NT004 [13], and OH3506 [14] as representative Japanese DS-1-like G1P [8] strains, which were analyzed in three independent studies in 2014.
segments (VP4, VP6, VP1-3, and NSP1-5) with genotypes identical to those of strains SKT-281, SKT-289, and LS-04. Green shading indicates the VP7 gene segments with a G3 genotype identical to those of strains SKT-281 and SKT-289. Blue shading indicates the VP7 gene segments with a G2 genotype identical to that of strain LS-04. "−" indicates that no sequence data were available in the DDBJ and EMBL/GenBank data libraries. a The gene segments that are most similar to those of strain SKT-281. b The gene segments that are most similar to those of strain SKT-289. c The gene segments that are most similar to those of strain LS-04.
In the present study, we analyzed the whole genomes of three DS-1-like intergenogroup reassortant strains having G3P [8] (strains SKT-281 and SKT-289) and G2P [8] (strain LS-04) genotypes identified in stool samples from hospitalized children with severe gastroenteritis in Thailand. No difference in clinical presentation and severity was found among RVA genotypes (Tacharoenmuang et al., in preparation). All the three strains showed unique genotype constellations comprising mixtures of genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 (strains SKT-281 and SKT-289) and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 (strain LS-04). With the exception of the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P [8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P [8] strains, whereas the remaining VP7 and NSP4 genes were assumed to be of equine and bovine origin, respectively. Therefore, strains SKT-281 and SKT-289 seemed to have been derived through reassortment event(s) between DS-1-like G1P [8], animal-like human and/or animal rotaviruses. However, the exact origins of the VP7 and NSP4 genes of strains SKT-281 and SKT-289 could not be ascertained due to a lack of a sufficient number of representative animal-like human and animal strains as references. Furthermore, strains SKT-281 and SKT-289 were very closely related to each other in all the 11 gene segments, indicating the derivation of the two strains from a common ancestor. In contrast, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) were assumed to have originated from locally circulating DS-1-like G2P [4] human rotaviruses, while three genes (VP4, VP2, and NSP1) appeared to be derived from DS-1-like G1P [8] strains. The remaining NSP2 gene of strain LS-04 was assumed to be of bovine origin, although the exact origin of this gene of strain LS-04 could not be ascertained due to a lack of a sufficient number of representative bovine-like human and bovine strains as references. Thus, strain LS-04 appeared to be a multiple reassortment strain involving DS-1-like G1P [8], locally circulating DS-1-like G2P [4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P [8] strains seemed to have been generated through reassortment involving human and animal strains.
Of note is that the VP4 gene of strain LS-04 showed the closest relationship with the cognate genes of locally circulating Wa-like G1P [8] strains, as well as Thai DS-1-like G1P [8] strains, being slightly away from the cluster comprising strains SKT-281 and SKT-289, and Japanese DS-1-like G1P [8] strains (Fig 6). These results might imply the occurrence of reassortment between Japanese DS-1-like G1P [8] and the locally circulating Wa-like G1P [8] strains to form Thai DS-1-like G1P [8] strains having the LS-04-like VP4 genes. Another possibility is that the DS-1-like G1P [8] strains might have originated from not a single, but at least two distinct ancestral Wa-like G1P [8] strains that possessed slightly different VP4 genes. However, global Dynamic Evolution of Emerging DS-1-Like G1P [8] Human Rotaviruses in Thailand rotavirus strain collection is required to determine the exact evolutionary patterns of DS-1-like G1P [8] strains and related reassortant ones. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P [8] and G2P [8] genotypes that have emerged in Thailand. We need to continue surveying the emergence of any unusual RVA strains such as DS-1-like G1P [8] strains and related reassortant ones in order to determine the relationship with the recent introduction of rotavirus vaccines.
The emergence of DS-1-like intergenogroup reassortant strains having G3P [8] and G2P [8] genotypes in Thailand may imply the constant circulation of DS-1-like G1P [8] strains and the occurrence of reassortment involving them, at least in Asia. Because DS-1-like G1P [8] strains have successfully spread in broad locations in Asia [13][14][15][16], continued surveillance of DS-1-like G1P [8] strains and related reassortant ones is required. Although most studies on RVA genotype distributions have been based on only G/P defining genes, PCR-based genotyping for non-G/P defining gene(s) or PAGE analysis would assist the identification of unusual RVA strains such as DS-1-like G1P [8] strains and related reassortant ones. Furthermore, whole genome-based analyses are essential to understand the evolutionary dynamics of emerging DS-1-like G1P [8] strains and related reassortant ones.