Whole Genomic Analysis of an Unusual Human G6P[14] Rotavirus Strain Isolated from a Child with Diarrhea in Thailand: Evidence for Bovine-To-Human Interspecies Transmission and Reassortment Events

An unusual rotavirus strain, SKT-27, with the G6P[14] genotypes (RVA/Human-wt/THA/SKT-27/2012/G6P[14]), was identified in a stool specimen from a hospitalized child aged eight months with severe diarrhea. In this study, we sequenced and characterized the complete genome of strain SKT-27. On whole genomic analysis, strain SKT-27 was found to have a unique genotype constellation: G6-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The non-G/P genotype constellation of this strain (I2-R2-C2-M2-A3-N2-T6-E2-H3) is commonly shared with rotavirus strains from artiodactyls such as cattle. Phylogenetic analysis indicated that nine of the 11 genes of strain SKT-27 (VP7, VP4, VP6, VP2-3, NSP1, NSP3-5) appeared to be of artiodactyl (likely bovine) origin, while the remaining VP1 and NSP2 genes were assumed to be of human origin. Thus, strain SKT-27 was found to have a bovine rotavirus genetic backbone, and thus is likely to be of bovine origin. Furthermore, strain SKT-27 appeared to be derived through interspecies transmission and reassortment events involving bovine and human rotavirus strains. Of note is that the VP7 gene of strain SKT-27 was located in G6 lineage-5 together with those of bovine rotavirus strains, away from the clusters comprising other G6P[14] strains in G6 lineages-2/6, suggesting the occurrence of independent bovine-to-human interspecies transmission events. To our knowledge, this is the first report on full genome-based characterization of human G6P[14] strains that have emerged in Southeast Asia. Our observations will provide important insights into the origin of G6P[14] strains, and into dynamic interactions between human and bovine rotavirus strains.


Introduction
Group A rotavirus (RVA), a member of the Reoviridae family, is the most important etiological agent of severe 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 per year in children <5 years of age [1]. More than half of these deaths were estimated to occur in developing countries in Asia and Africa [1,2].

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 sequence was determined for strain SKT-27, which was identified as the sole pathogen causing diarrhea in stool specimens from a hospitalized child aged eight months with severe diarrhea in Sukhothai Province in November 2012 during the RVA strain surveillance program in Phetchaboon and Sukhothai Provinces, Thailand in 2012-2014, which involved a total of 685 RVA-positive fecal samples (Tacharoenmuang et al., in preparation). Stool specimens were collected from hospital-based surveillance activities under the RVA vaccine effectiveness evaluation study in Thailand. Stool samples containing strain SKT-27 were kept at −30°C until use.

Viral dsRNA extraction
The viral dsRNAs were extracted from stool 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 [32] to determine the genomic dsRNA profile. 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 [30,33]. Briefly, a 200 bp fragment library ligated with bar-coded adapters was constructed for strain SKT-27 using an NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (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 performed 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 strain SKT-27. The nucleotide sequences were translated into amino acid sequences using GENETYX v11 (GENETYX).

Determination of RVA genotypes
The genotype of each of the 11 gene segments of strain SKT-27 was determined using the RotaC v2.0 automated genotyping tool (http://rotac.regatools.be/) [34] according to the guidelines proposed by the Rotavirus Classification Working Group (RCWG).

Phylogenetic analyses
Sequence comparisons were performed as described previously [33,35]. Briefly, multiple alignment of each viral gene was performed using CLUSTAL W [36]. Phylogenetic trees were constructed using the maximum likelihood method and the Tamura-Nei substitution model using MEGA6.06 [37]. The reliability of the branching order was estimated from 1000 bootstrap replicates [38]. The results of phylogenetic analyses were validated using several other genetic distance models, such as the Jukes-Cantor, Kimura 2-parameter, Tamura 3-parameter, and Hasegawa-Kishino-Yano ones (data not shown).

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 NSP1-5/6 genes of strain SKT-27 are LC055547-LC055557, respectively.

Results and Discussion
Extraction of genomic dsRNAs of strain SKT-27 Virion dsRNAs of strain SKT-27 were extracted from stool specimens and then analyzed by PAGE. Fig 1 shows  Nucleotide sequencing and whole-genome-based genotyping of strain SKT-27 PCR-based genotyping [41] showed that strain SKT-27 has the G6 genotype (Tacharoenmuang et al., in preparation). Because G6 strains have been detected almost exclusively in artiodactyls such as cattle [6,18], the detection of a G6 strain in the human population was suggestive of zoonotic transmission. The whole genome of strain SKT-27 was amplified using a sequenceindependent primer set and then sequenced successfully. Illumina MiSeq sequencing yielded 6.8 x 10 5 reads (~145 bp average length) for strain SKT-27. Complete or nearly complete nucleotide sequences of all the 11 gene segments of strain SKT-27 could be obtained.

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 [29].
The NSP1 gene of strain SKT-27 exhibited the highest nucleotide sequence identity (93.7%) with the cognate gene of Irish bovine-like giraffe strain GirRV (G10P[11]) (Fig 2). On phylogenetic analysis, strain SKT-27 was shown to be closely related with strain GirRV near the clusters formed by several bovine and bovine-like strains (Fig 9).
In the present study, we analyzed the whole genome of a Thai strain, SKT-27, with the G6P [14] genotypes (RVA/Human-wt/THA/SKT-27/2012/G6P [14]) from a child with acute gastroenteritis. Strain SKT-27 showed a unique genotype constellation: G6-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The non-G/P genotype constellation of this strain (I2-R2-C2-M2-A3-N2-a The gene segments that are most similar to those of strain SKT-27. "−" indicates that no sequence data were available in the DDBJ and EMBL/GenBank data libraries. b Genotype assignment of strain DK11601 based on a report by Midgley et al. (2012). To our knowledge, to date, nucleotide sequence accession numbers for the VP7 and VP4 genes of strain DK11601 are not available in the DDBJ and EMBL/GenBank data libraries.
However, the exact origins of the VP1 and NSP2 genes of strain SKT-27 could not be ascertained due to a lack of a sufficient number of representative strains as references. Therefore, we could not determine if the suspected ancestral G6P [14] strain of strain SKT-27 had human-like VP1 and NSP2 genes in its genome before it was transmitted from cattle to humans. In any case, strain SKT-27 was assumed to be derived through interspecies transmission and/or reassortment events involving bovine and human RVA strains.
Notably, the VP7 gene of strain SKT-27 was located in G6 lineage-5, away from the clusters comprising other G6P [14] strains in G6 lineages-2/6. This could support the hypothesis that distinct human G6P [14] strains are more likely the result of individual interspecies transmissions from artiodactyls rather than the result of human-to-human transmission after interspecies transmission [18,20]. To our knowledge, this is the first description of full genome-based characterization of human G6P [14] strains that have emerged in Southeast Asia.
Our findings add to the increasing evidence supporting animal-to-human interspecies transmission and reassortment events. The bovine origin of strain SKT-27 also suggests interspecies transmission due to the close proximity of humans to livestock, especially in developing countries in Asia where there is intimate contact between humans and livestock such as cattle. Because G6P [14] strains have been able to cause constant human infections worldwide, continuing RVA surveillance of this G-P combination in the human population may be required. Simultaneous monitoring of RVA strains in humans and animals is also essential for a better understanding of RVA ecology. Furthermore, whole genome-based analyses are essential to understand the evolutionary dynamics of novel RVA strains such as G6P [14] strains.