Emergence and Characterization of Unusual DS-1-Like G1P[8] Rotavirus Strains in Children with Diarrhea in Thailand

The emergence and rapid spread of unusual DS-1-like G1P[8] rotaviruses in Japan have been recently reported. During rotavirus surveillance in Thailand, three DS-1-like G1P[8] strains (RVA/Human-wt/THA/PCB-180/2013/G1P[8], RVA/Human-wt/THA/SKT-109/2013/G1P[8], and RVA/Human-wt/THA/SSKT-41/2013/G1P[8]) were identified in stool specimens from hospitalized children with severe diarrhea. In this study, we sequenced and characterized the complete genomes of strains PCB-180, SKT-109, and SSKT-41. On whole genomic analysis, all three strains exhibited a unique genotype constellation including both genogroup 1 and 2 genes: G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. This novel genotype constellation is shared with Japanese DS-1-like G1P[8] strains. Phylogenetic analysis revealed that the G/P genes of strains PCB-180, SKT-109, and SSKT-41 appeared to have originated from human Wa-like G1P[8] strains. On the other hand, the non-G/P genes of the three strains were assumed to have originated from human DS-1-like strains. Thus, strains PCB-180, SKT-109, and SSKT-41 appeared to be derived through reassortment event(s) between Wa-like G1P[8] and DS-1-like human rotaviruses. Furthermore, strains PCB-180, SKT-109, and SSKT-41 were found to have the 11-segment genome almost indistinguishable from one another in their nucleotide sequences and phylogenetic lineages, indicating the derivation of the three strains from a common origin. Moreover, all the 11 genes of the three strains were closely related to those of Japanese DS-1-like G1P[8] strains. Therefore, DS-1-like G1P[8] strains that have emerged in Thailand and Japan were assumed to have originated from a recent common ancestor. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like G1P[8] strains that have emerged in an area other than Japan. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] rotaviruses.


Introduction
Group A rotavirus (RVA), a member of the Reoviridae family, is the most important etiological agent of severe gastroenteritis in infants and young children. RVA infections are associated with high morbidity and mortality, being responsible for an estimated 453,000 deaths each year in children <5 years of age [1]. The RVA virion is a non-enveloped, triple-layered icosahedron that encapsidates an 11-segment genome of double-stranded (ds)RNA [2]. Due to the segmented nature of the genome, reassortment between/among RVAs is one of the major processes of genetic evolution of this medically important virus.
Whole genome-based analysis is a reliable method for obtaining conclusive data on the origin of an RVA strain, and for tracing its evolutionary pattern [7,14]. To date, the whole genome sequences of several Japanese DS-1-like G1P [8] strains have been fully sequenced and characterized, which indicated the occurrence of reassortment event(s) between Wa-like G1P [8] and DS-1-like human rotaviruses [10,13]. Furthermore, Japanese DS-1-like G1P [8] strains were very closely related to one another in all the 11 genes, indicating that these Japanese strains are epidemiologically linked to one another [10]. However, as the overall genomic constellation and the genomic diversity of DS-1-like G1P [8] strains remain to be elucidated, whole genomic analysis of the Thai DS-1-like G1P [8] strains might be useful for obtaining a more precise understanding of the evolutionary pattern of DS-1-like G1P [8] strains. In this study, deep sequencing with the next generation sequencing (NGS) Illumina MiSeq platform was performed to obtain the complete nucleotide sequences of the whole genomes of these three Thai DS-1-like G1P [8] strains.

Ethics statement
The study was approved by the Ethical Review Committee for Research in 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 PCB-180, SKT-109, and SSKT-41, which were identified in three fecal specimens from hospitalized children aged 16

Viral dsRNA extraction
The viral dsRNAs were extracted from fecal suspensions using a QIAamp Viral RNA Mini Kit (Qiagen). The extracted dsRNAs were used for (i) polyacrylamide gel electrophoresis (PAGE) analysis, and (ii) whole genomic analysis. For PAGE analysis, the dsRNAs were electrophoresed in a 10% polyacrylamide gel for 16 h at 20 mA at room temperature, followed by silver staining [15] to determine the genomic dsRNA profile. For whole genomic analysis, viral dsRNAs were subjected to Illumina MiSeq sequencing as described below.

Phylogenetic analyses
Sequence comparisons were carried out as described previously [17,20]. Briefly, multiple alignment of each viral gene was performed using CLUSTAL W [21]. Phylogenetic trees were constructed using the maximum likelihood method and the Tamura-Nei substitution model using MEGA6.06 [22]. The reliability of the branching order was estimated from 1000 bootstrap replicates [23].

Nucleotide sequence accession numbers
The nucleotide sequence data presented in this manuscript have been deposited in the DDBJ and EMBL/GenBank data libraries. The accession numbers for the nucleotide sequences of the VP1-4, VP6-7, and NSP1-5 genes of strains PCB-180, SKT-109, and SSKT-41 are LC066639-LC066649, LC066650-LC066660, and LC066661-LC066671, respectively.

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 [7]. In this study, we employed strains HC12016 [13], NT004 [10], and OH3506 [12] as representative Japanese DS-1-like G1P [8] strains, which were analyzed in three independent studies in 2014.
In the present study, we analyzed the whole genomes of three DS-1-like G1P [8] strains that have emerged in Thailand (strains PCB-180, SKT-109, and SSKT-41) identified in stool specimens from hospitalized children with severe gastroenteritis during the RVA surveillance program, which involved a total of 687 RVA-positive stool samples. No difference in clinical presentation and severity was found among genotypes (Tacharoenmuang et al., in preparation). All the three Thai DS-1-like G1P [8] strains showed a unique genotype constellation including both genogroup 1 and 2 genes: G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. This unusual genotype constellation is shared with Japanese DS-1-like G1P [8] strains. Phylogenetic analysis revealed that the outer capsid genes (VP7 and VP4) of strains PCB-180, SKT-109, and SSKT-41 appeared to have originated from human Wa-like G1P [8] strains. On the other hand, the inner capsid and nonstructural genes (VP6, VP1-3, and NSP1-5) of these three strains were assumed to have originated from human DS-1-like strains. Therefore, strains PCB-180, SKT-109, and SSKT-41 were assumed to have been derived through reassortment event(s) between Wa-like G1P [8] and DS-1-like human rotaviruses. Furthermore, on phylogenetic analysis, strains PCB-180, SKT-109, and SSKT-41 were found to be very closely related to one another as to all the 11 genes, indicating the derivation of the three strains from a common origin. Moreover, all the 11 genes of these three strains were closely related with those of Japanese DS-1-like G1P [8] strains. Thus, DS-1-like G1P [8] strains that have emerged in Thailand and Japan appeared to have a common origin.
Notably, the VP4 genes of strains PCB-180, SKT-109, and SSKT-41 were closely related to those of Wa-like G1P [8] strains isolated in Thailand or the United States in 2007-2011. This could support the hypothesis that the reassortment event(s) between Wa-like G1P [8] and DS-1-like human strains occurred outside Japan, and the resultant DS-1-like G1P [8] strain was somehow introduced into Japan and then spread throughout the entire nation [10]. However, included in these vaccines, continuing RVA surveillance of DS-1-like G1P [8] strains is required. Although most studies on RVA genotype distributions have been focused on only G/ P genes, PCR-based genotyping for non-G/P gene(s) or PAGE analysis should be performed to detect unusual RVA strains such as DS-1-like G1P [8] ones. Furthermore, whole genome-based analyses are essential to understand the evolutionary dynamics of emerging DS-1-like G1P [8] strains.