ABSTRACT
This study reports the coding-complete genome sequences of three rotavirus A (RVA) reference strains previously adapted in tissue culture: RVA/Mouse-tc/USA/EDIM/XXXX/G16P[16] with a G16-P[16]-I7-R7-C7-M8-A7-N7-T10-E7-H9 genotype constellation, RVA/Human-tc/USA/Ph158/1998/G9P[6] with a G9-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genotype constellation, and RVA/Human-tc/USA/CC425/1998/G3P[9] with a G3-P[9]-I2-R2-C2-M2-A3-N2-T1-E2-H3 genotype constellation.
KEYWORDS: rotavirus, next-generation sequencing, EDIM, Ph158, CC425
ANNOUNCEMENT
Group A rotaviruses (RVAs) cause acute gastroenteritis in young children and animals (1 – 3). Belonging to the Sedoreoviridae family, RVA genomes consist of 11 double‐stranded RNA (dsRNA) segments whose classification is based on sequencing of all genome segments (4, 5). The genotype constellation for the 11 RVA segments is Gx‐P[x]‐Ix‐Rx‐Cx‐Mx‐Ax‐Nx‐Tx‐Ex‐Hx for genes VP7‐VP4‐VP6‐VP1‐VP2‐VP3‐NSP1‐NSP2‐NSP3‐NSP4‐NSP5/6, respectively (6). Here, we report the coding-complete genome sequences for reference strains RVA/Mouse-tc/USA/EDIM/XXXX/G16P[16] (referred to as EDIM), RVA/Human-tc/USA/Ph158/1998/G9P[6] (Ph158), and RVA/Human-tc/USA/CC425/1998/G3P[9] (CC425), which were previously partially characterized as follows: EDIM as G16-P[16]-I7-Rx-Cx-Mx-A7-Nx-Tx-E7-Hx (7), Ph158 as G9-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx (8), and CC425 as G3-P[9]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-E2-Hx (9).
EDIM was initially isolated from the stool of an infected mouse and adapted for mouse models of human rotavirus infection (10). Ph158 was cultured to help characterize human G9 strains (8), and CC425 was cultured for P[9] characterization (9). These reference strains have significantly impacted the field through their use in early studies evaluating the immunological mechanisms against RVA, phylogenetic analyses of emerging strains, and assay development for genotyping; however, the gene-coding regions have yet to be fully sequenced (10, 11). In this report, next-generation sequencing (NGS) was performed to facilitate accurate interpretations of the origin of the reference strains and assist in tracing their evolutionary patterns.
Each strain was propagated in MA104 cell monolayers from existing stocks, as previously described (12). Viral stocks were maintained in IMDM media, and undiluted supernatant was used for the dsRNA extraction. The NGS methods were performed as previously described (13). Briefly, RVA dsRNA was extracted using the MagNA Pure Compact RNA Isolation Kit (Roche). The dsRNA served as a template for the cDNA sequencing library using the NEBNext Ultra RNA Library Prep Kit and Multiplex Oligos for Illumina Kit (New England Biolabs). The library was sequenced using a MiSeq Reagent Kit v.2 (500-cycle) on a MiSeq System (Illumina).
The total number of paired-end reads was 1,267,346 for EDIM, 1,064,027 for Ph158, and 1,486,484 for CC425. The average read length ranged from 189 to 198 bp. Reference-guided assembly was performed using CLC Genomics Workbench 20.0.3 software (14), resulting in coding-complete sequences for all segments (Table 1). To determine the open reading frames (ORFs), study sequences were cross-referenced using additional sequences with well-established ORFs from GenBank and further validated using established literature that elucidates the conventionally recognized gene-coding assignments (15, 16). Partial non-coding sequences were reported depending on coverage. Genotypes were determined according to the guidelines of the Rotavirus Classification Working Group (7) using NCBI’s BLASTN tool (17).
TABLE 1.
Sequence details and BLASTN results of reference strains EDIM, Ph158, and CC425
| Data for top BLASTN match | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Strain | Gene | Genotype | GC content (%) | Full sequence length (bp) | Coding sequence length (bp) | ORF | GenBank accession no. | BLASTN description | Identity (%) | GenBank accession no. |
| EDIM | VP7 | G16 | 39.6 | 1062 | 981 | Complete | MT276814 | Murine rotavirus strain ETD_822 VP7 gene, complete cds | 99.8 | GQ479955.1 |
| VP4 | P[16] | 45.3 | 2347 | 2325 | Complete | MT276815 | Murine rotavirus EW outer capsid protein VP4 gene, complete cds | 100.0 | U08429.1 | |
| VP6 | I7 | 43.7 | 1355 | 1194 | Complete | MT276816 | Murine rotavirus strain ETD_822 VP6 gene, complete cds | 100.0 | GQ479952.1 | |
| VP1 | R7 | 39.7 | 3294 | 3267 | Complete | MT276817 | Murine rotavirus strain ETD_822 VP1 gene, complete cds | 99.9 | GQ479947.1 | |
| VP2 | C7 | 40.7 | 2667 | 2637 | Complete | MT276818 | Murine rotavirus strain ETD_822 VP2 gene, complete cds | 99.8 | GQ479948.1 | |
| VP3 | M8 | 39.2 | 2583 | 2508 | Complete | MT276819 | Murine rotavirus strain ETD_822 VP3 gene, complete cds | 99.9 | GQ479949.1 | |
| NSP1 | A7 | 41.5 | 1599 | 1482 | Complete | MT276820 | Murine rotavirus strain ETD_822 NSP1 gene, complete cds | 99.4 | GQ479951.1 | |
| NSP2 | N7 | 39.9 | 1043 | 951 | Complete | MT276821 | Murine rotavirus strain ETD_822 NSP2 gene, complete cds | 99.8 | GQ479954.1 | |
| NSP3 | T10 | 41.5 | 1069 | 933 | Complete | MT276822 | Murine rotavirus strain ETD_822 NSP3 gene, complete cds | 99.7 | GQ479953.1 | |
| NSP4 | E7 | 43.6 | 740 | 528 | Complete | MT276823 | Murine rotavirus strain ETD_822 NSP4 gene, complete cds | 99.6 | GQ479956.1 | |
| NSP5 | H9 | 42.3 | 647 | 594 | Complete | MT276824 | Murine rotavirus strain ETD_822 NSP5/6 gene, complete cds | 99.9 | GQ479957.1 | |
| Ph158 | VP7 | G9 | 34.3 | 1027 | 981 | Complete | MT339194 | Rotavirus G9 VP7 gene for neutralizing capsid protein, strain PH158, genomic RNA | 99.9 | AJ491183.1 |
| VP4 | P[6] | 35.4 | 2347 | 2328 | Complete | MT339195 | Human rotavirus G9P[6] strain GR 10924/99 VP4 gene, complete cds | 99.6 | FJ183356.1 | |
| VP6 | I2 | 37.0 | 1285 | 1194 | Complete | MT339196 | Rotavirus A strain RVA/Human-wt/UGA/MSK-13–048/2013 /G9P[8] VP6 (VP6) mRNA, complete cds | 99.7 | KX655510.1 | |
| VP1 | R2 | 32.4 | 3273 | 3267 | Complete | MT339197 | Human rotavirus G9P[6] strain GR 10924/99 VP1 gene, complete cds | 99.5 | FJ183353.1 | |
| VP2 | C2 | 32.9 | 2654 | 2640 | Complete | MT339198 | Rotavirus A strain MRC-DPRU9317 segment 2, complete sequence | 99.4 | JN605438.1 | |
| VP3 | M2 | 30.8 | 2571 | 2508 | Complete | MT339199 | Human rotavirus G9P[6] strain GR 10924/99 VP3 gene, complete cds | 99.5 | FJ183355.1 | |
| NSP1 | A2 | 27.8 | 1536 | 1461 | Complete | MT339200 | Rotavirus A strain RVA/Human-wt/UGA/MSK-13–048/2013 /G9P[8] NSP1 (NSP1) mRNA, complete cds | 99.4 | KX655512.1 | |
| NSP2 | N2 | 35.8 | 1032 | 954 | Complete | MT339201 | Human rotavirus G9P[6] strain GR 10924/99 NSP2 gene, complete cds | 99.7 | FJ183361.1 | |
| NSP3 | T2 | 31.6 | 1044 | 933 | Complete | MT339202 | Rotavirus A strain MRC-DPRU9317 segment 7, complete sequence | 99.6 | JN605445.1 | |
| NSP4 | E2 | 38.5 | 719 | 528 | Complete | MT339203 | Human rotavirus NSP4 gene for nonstructural protein 4, strain 1071 | 100.0 | AJ236754.1 | |
| NSP5 | H2 | 30.7 | 716 | 603 | Complete | MT339204 | Rotavirus A strain RVA/Human-wt/ZAF/MRC-DPRU9164/1999 /G9P[6] segment 11 nonstructural protein 5 (NSP5) and nonstructural protein 6 (NSP6) genes, complete cds | 99.6 | KJ752248.1 | |
| CC425 | VP7 | G3 | 35.8 | 1054 | 981 | Complete | MT276803 | Human rotavirus VP7 gene for VP7 capsid protein, genomic RNA, strain CC425, P3[9],G3 | 100.00 | AJ311738.1 |
| VP4 | P[9] | 35.9 | 2355 | 2328 | Complete | MT276804 | Rotavirus A strain RVA/Human-tc/USA/Se584/1998 /G6P[9] outer capsid protein VP4 gene, complete cds | 98.03 | EF672605.1 | |
| VP6 | I2 | 38.4 | 1351 | 1194 | Complete | MT276805 | Equine rotavirus A strain RVA/Horse-wt/IND/ERV2/2015 /G6P[1] segment 6 VP6 gene, complete cds | 97.71 | OK651101.1 | |
| VP1 | R2 | 34.8 | 3292 | 3267 | Complete | MT276806 | Bovine rotavirus core protein (VP1) gene, complete cds | 97.17 | J04346.1 | |
| VP2 | C2 | 35.2 | 2669 | 2643 | Complete | MT276807 | Rotavirus A strain RVA/Human-tc/USA/Se584/1998 /G6P[9] core shell protein VP2 gene, complete cds | 98.75 | EF583042.1 | |
| CC425 | VP3 | M2 | 32.4 | 2588 | 2508 | Complete | MT276808 | Rotavirus A strain RVA/Cow-tc/USA/NCDV/1967 /G6P6[1] VP3 gene, complete cds | 97.33 | DQ870495.1 |
| NSP1 | A3 | 35.7 | 1570 | 1476 | Complete | MT276809 | Rotavirus A strain RVA/Human-tc/USA/Se584/1998 /G6P[9] nonstructural protein one gene, complete cds | 98.58 | EF672606.1 | |
| NSP2 | N2 | 35.0 | 1049 | 954 | Complete | MT276810 | Rotavirus A strain RVA/Human-tc/USA/Se584/1998 /G6P[9] nonstructural protein two gene, complete cds | 99.27 | EF672608.1 | |
| NSP3 | T1 | 33.7 | 1074 | 933 | Complete | MT276811 | Rotavirus RVA/Human-wt/USA/12US1134/2012 /G3P[9] NSP3 (NSP3) gene, complete cds | 98.7 | KF500516.1 | |
| NSP4 | E2 | 41.4 | 740 | 528 | Complete | MT276812 | Human Rotavirus NSP4 gene for nonstructural-protein 4, genomic RNA, strain CC425, P3[9],G3 | 100.0 | AJ311728.1 | |
| NSP5 | H3 | 38.8 | 641 | 597 | Complete | MT276813 | Rotavirus A strain RVA/Human-wt/ITA/PAI58/1996 /G3P[9] NSP5 gene, complete cds | 98.4 | GU296419.1 | |
NGS and genotyping results are presented in Table 1. The genome constellations were determined to be: EDIM as G16-P[16]-I7-R7-C7-M8-A7-N7-T10-E7-H9, Ph158 as G9-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2, and CC425 as G3-P[9]-I2-R2-C2-M2-A3-N2-T1-E2-H3. The constellation for EDIM has been observed in RVA strains of murine origin and was most closely related to murine strain ETD_822 (6). Ph158 was found to possess a complete genogroup 2 constellation. CC425 is closely related to equine, bovine, and human-origin strains, including G6P[9] strain Se584 (18). While further analysis is needed, this report presents the complete gene sequences of common reference strains used throughout RVA research.
ACKNOWLEDGMENTS
Support for this study was provided by CDC program funding.
The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Names of specific vendors, manufacturers, or products are included for public health and informational purposes; inclusion does not imply endorsement of the vendors, manufacturers, or products by the Centers for Disease Control and Prevention or the U.S. Department of Health and Human Services.
Contributor Information
Mary C. Casey-Moore, Email: quc2@cdc.gov.
Jelle Matthijnssens, Katholieke Universiteit Leuven, Leuven, Belgium .
DATA AVAILABILITY
Project information, including data sets and the raw sequence reads, has been deposited in BioProject under accession no. PRJNA614927. NCBI GenBank accession numbers are as follows: EDIM (MT276814-MT276824), PH158 (MT339194-MT339204), and CC425 (MT276803- MT276813). Accession numbers for individual genes are listed in Table 1.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Project information, including data sets and the raw sequence reads, has been deposited in BioProject under accession no. PRJNA614927. NCBI GenBank accession numbers are as follows: EDIM (MT276814-MT276824), PH158 (MT339194-MT339204), and CC425 (MT276803- MT276813). Accession numbers for individual genes are listed in Table 1.