Full genome characterization of novel DS-1-like G9P[8] rotavirus strains that have emerged in Thailand

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotaviruses having G1/3/8 genotypes have been recently reported from major parts of the world (Africa, Asia, Australia, Europe, and the Americas). During rotavirus surveillance in Thailand, three novel intergenogroup reassortant strains possessing the G9P[8] genotype (DBM2017-016, DBM2017-203, and DBM2018-291) were identified in three stool specimens from diarrheic children. In the present study, we determined and analyzed the full genomes of these three strains. On full-genomic analysis, all three strains were found to share a unique genotype constellation comprising both genogroup 1 and 2 genes: G9-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis demonstrated that each of the 11 genes of the three strains was closely related to that of emerging DS-1-like intergenogroup reassortant, human, and/or locally circulating human strains. Thus, the three strains were suggested to be multiple reassortants that had acquired the G9-VP7 genes from co-circulating Wa-like G9P[8] rotaviruses in the genetic background of DS-1-like intergenogroup reassortant (likely equine-like G3P[8]) strains. To our knowledge, this is the first description of emerging DS-1-like intergenogroup reassortant strains having the G9P[8] genotype. Our observations will add to the growing insights into the dynamic evolution of emerging DS-1-like intergenogroup reassortant rotaviruses through reassortment.

Introduction Group A rotavirus (RVA) within the Reoviridae family, is the primary pathogen that causes severe gastroenteritis in young children and animals worldwide. RVA disease is responsible for an estimated 128,500-215,000 deaths among children <5 years of age annually [1,2]. The RVA genome consists of 11 segments of double-stranded (ds)RNA, encoding six structural proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP6) [3]. The segmented nature of the genome facilitates reassortment between/among RVA strains, and the reassortment plays one of the major roles in the dynamic evolution of RVAs [4].
In 2017-2018, we detected five novel DS-1-like intergenogroup reassortant strains having the G9P [8] genotype with a short electropherotype in diarrheic children in Thailand, a total of 429 RVA-positive stool specimens being examined by RT-PCR-based G/P genotyping and polyacrylamide gel electrophoresis (PAGE) analysis during the RVA surveillance in 2015-2018 (Tacharoenmuang et al., in preparation), while no DS-1-like G9P [8] strain was detected in 2015-2016. Because these DS-1-like G9P [8] strains were unusual, full-genomic analysis of these Thai strains might be useful for obtaining a more precise understanding of the evolutionary dynamics of emerging DS-1-like intergenogroup reassortant strains. In the present study, we sequenced and characterized the full genomes of three representative DS-1-like G9P [8] strains that have emerged in Thailand.

Ethics statement
This study was approved by the Ethical Review Committee for Research on Human Subjects, Ministry of Public Health, Thailand (Ref. no. 0032/2556). In this study, written informed consent for the testing of stool specimens for RVAs and characterization of detected RVA strains was obtained from the children's parents/guardians. Questionnaire information was deidentified and re-coded so that no information could be linked to any individual participant.

Virus strains
During the RVA surveillance program in Thailand in 2015-2018, which involved a total of 429 RVA-positive fecal specimens (Tacharoenmuang et al., in preparation), five G9P [8] strains with a short electropherotype were detected in stool samples from diarrheic children (aged 5 months to 5 years 5 months) admitted to Bhumibol Adulyadej Hospital in Bangkok. Out of the five identified G9P [8] strains with a short electropherotype, three representative strains showing intense genomic dsRNA bands on PAGE analysis were selected (strains DBM2017-016 and DBM2017-203 in 2017, and strain DBM2018-291 in 2018) for full genome-based analysis. In addition, the nucleotide sequences of the full genomes of five locally circulating HuRVA strains (three G2P [4] strains with a short electropherotype (DBM2017-003, DBM2017-015, and DBM2018-105) and two G9P [8] strains with a long electropherotype (DBM2017-014 and DBM2018-111)) detected in stool specimens from diarrheic children (aged 1 year 7 months to 10 years 5 months) admitted to Bhumibol Adulyadej Hospital were determined as well, as references. Stool samples containing the above-mentioned eight HuRVA strains were kept at −30˚C until use.

Viral genomic dsRNA extraction, cDNA library building, and Illumina MiSeq sequencing
RVA genomic dsRNAs were extracted from stool specimens using a QIAamp Viral RNA Mini Kit (Qiagen), and the dsRNAs were subjected to Illumina MiSeq sequencing as described previously [35,36]. In brief, a 200 bp fragment library ligated with bar-coded adapters was built for the eight HuRVA strains using an NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (New England Biolabs), and NEBNext Multiplex Oligos for Illumina (New England Biolabs) according to the manufacturer's instructions. The cDNA library was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). After assessing the quality and quantity of the purified cDNA library, nucleotide sequencing was performed on an Illumina MiSeq sequencer (Illumina) using a MiSeq Reagent Kit v2 (Illumina) to generate 151 paired-end reads. Bioinformatics analysis was carried out according to the protocol previously described [37]. Sequence reads were trimmed to exclude the adapters, primers, and low-quality sequences, using CLC Genomics Workbench v8.0.1 (CLC Bio). The parameter settings for the quality trimming were as follows: trim using quality scores, limit = 0.08; trim ambiguous nucleotides, maximum number of ambiguities = 4; and filter on length, discard reads below length = 15. Data analysis was performed using CLC Genomics Workbench v8.0.1. Contigs were assembled from the obtained sequence reads (trimmed) by de novo assembly. Using the assembled contigs as query sequences, the Basic Local Alignment Search Tool (BLAST) nonredundant nucleotide database was searched to determine which contig represents the full-length nucleotide sequence of each gene segment of the eight HuRVA strains. To further refine the contigs, the sequence reads of each gene were mapped back to the assembled contigs. The nucleotide sequences were translated into amino acid sequences using GENETYX v11 (GENE-TYX, Tokyo, Japan).

Phylogenetic analyses
The three study strains, DBM2017-016, DBM2017-203, and DBM2018-291, were further characterized by constructing phylogenetic trees using the full-length sequences for each of the 11 genes because phylogenetic analysis of RVA nucleotide sequences provides precise information on the origin of a given strain, and for tracing its evolutionary pattern, even within the same genotype [5,41] (Fig 2A-2K). The nucleotide sequence identities between the three study strains and a representative close strain as to each gene are shown in Table 1.
The decreasing detection of common HuRVA strains such as Wa-like G1P [8] viruses and increasing detection of uncommon HuRVA strains such as DS-1-like intergenogroup reassortant viruses are an emerging concern in relation to HuRVA vaccine strategies. Although it is apparent that the current live-attenuated HuRVA vaccines (Rotarix (GlaxoSmithKline) and RotaTeq (Merck)) are highly effective against severe HuRVA disease, these vaccines might have put selective pressure on circulating HuRVA strains [6,12,46,47]. HuRVA vaccines have not been introduced to the national immunization program in Thailand yet, but a monovalent Rotarix vaccine (G1P [8]) was introduced in Sukhothai province as a pilot study in 2011, in which the vaccine effectiveness for hospitalized RVA diarrhea was 88% (95%CI 76-94) [48]. Thus, it would be important to consider monitoring the evolution and circulation of emergent DS-1-like intergenogroup reassortant strains in the context of vaccination after the introduction of vaccines to the national immunization program. It would be important to perform RT-PCR-based genotyping for non-G/P gene(s) or PAGE in addition to genotyping for G/P genes for detection of unusual intergenogroup reassortant HuRVAs including the studied DS-1-like G9P [8] strains.