Development and evaluation of an ARTIC-based amplicon sequencing assay for whole-genome characterization of respiratory syncytial virus
- 1Georgia Public Health Laboratory, Georgia Department of Public Health, Decatur, Georgia, USA
Abstract
Respiratory syncytial virus (RSV), a ~15.2 kb negative-sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV-A and RSV-B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 de-identified remnant clinical specimens (102 RSV-A and 112 RSV-B) with RT-PCR Cq values <31 were included. RSV genomes were amplified using ARTIC-style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV-A (median 53,433×; mean 51,076×) than RSV-B (median 49,699×; mean 46,945×), whereas genomic coverage was slightly lower for RSV-A (median 97.5%; mean 96.6%) than RSV-B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV-A and B.D.E.1 for RSV-B. For RSV-A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra- and inter-run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0–5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. Limits of detection were 4.4 TCID50/mL for RSV-A and 18.6 TCID50/mL for RSV-B. These results demonstrate that the ARTIC-based RSV WGS assay enables near real-time surveillance and strengthens data-driven public health responses to future outbreaks.
IMPORTANCE: RSV, with two major subtypes, RSV-A and RSV-B, causes acute respiratory infections that can be severe in infants under 6 months and older adults. Current RSV surveillance at the GPHL relies on the Thermo Fisher TaqMan Gene Expression Capillary assay, which detects and subtypes RSV but lacks resolution for lineage classification and identification of emerging variants. To address this critical gap, GPHL developed and evaluated an amplicon-based WGS assay using 214 de-identified RSV clinical specimens. Genomes were amplified using ARTIC-style and custom-primer sets, with ARTIC primers showing superior performance. The assay demonstrated strong sequencing depth, genomic coverage, specificity, repeatability, reproducibility, and low limits of detection. RSV lineages were accurately determined based on genetic variation. These results establish that the ARTIC-based WGS assay enables near real-time genomic surveillance, supporting monitoring of circulating RSV strains and informing data-driven public health responses.
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BibTeX
@article{smith2026development,
title = {{Development and evaluation of an ARTIC-based amplicon sequencing assay for whole-genome characterization of respiratory syncytial virus}},
author = {Keri Smith and James Martinez and Harrison Yu and Jason Harrison and Caleb Umunna and Blake Bertrand and Michael Heck and Ellen N. Kersh and Nandhakumar Balakrishnan and Tonia Parrott and Arunachalam Ramaiah},
journal = {Journal of Clinical Microbiology},
year = {2026},
month = sep,
doi = {10.1128/jcm.00468-26},
url = {https://doi.org/10.1128/jcm.00468-26},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Development and evaluation of an ARTIC-based amplicon sequencing assay for whole-genome characterization of respiratory syncytial virus
AU - Keri Smith
AU - James Martinez
AU - Harrison Yu
AU - Jason Harrison
AU - Caleb Umunna
AU - Blake Bertrand
AU - Michael Heck
AU - Ellen N. Kersh
AU - Nandhakumar Balakrishnan
AU - Tonia Parrott
AU - Arunachalam Ramaiah
PY - 2026
DA - 2026/09/03
JO - Journal of Clinical Microbiology
DO - 10.1128/jcm.00468-26
UR - https://doi.org/10.1128/jcm.00468-26
AB - Respiratory syncytial virus (RSV), a ~15.2 kb negative-sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV-A and RSV-B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 de-identified remnant clinical specimens (102 RSV-A and 112 RSV-B) with RT-PCR Cq values <31 were included. RSV genomes were amplified using ARTIC-style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV-A (median 53,433×; mean 51,076×) than RSV-B (median 49,699×; mean 46,945×), whereas genomic coverage was slightly lower for RSV-A (median 97.5%; mean 96.6%) than RSV-B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV-A and B.D.E.1 for RSV-B. For RSV-A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra- and inter-run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0–5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. Limits of detection were 4.4 TCID50/mL for RSV-A and 18.6 TCID50/mL for RSV-B. These results demonstrate that the ARTIC-based RSV WGS assay enables near real-time surveillance and strengthens data-driven public health responses to future outbreaks. IMPORTANCE: RSV, with two major subtypes, RSV-A and RSV-B, causes acute respiratory infections that can be severe in infants under 6 months and older adults. Current RSV surveillance at the GPHL relies on the Thermo Fisher TaqMan Gene Expression Capillary assay, which detects and subtypes RSV but lacks resolution for lineage classification and identification of emerging variants. To address this critical gap, GPHL developed and evaluated an amplicon-based WGS assay using 214 de-identified RSV clinical specimens. Genomes were amplified using ARTIC-style and custom-primer sets, with ARTIC primers showing superior performance. The assay demonstrated strong sequencing depth, genomic coverage, specificity, repeatability, reproducibility, and low limits of detection. RSV lineages were accurately determined based on genetic variation. These results establish that the ARTIC-based WGS assay enables near real-time genomic surveillance, supporting monitoring of circulating RSV strains and informing data-driven public health responses.
SN - 0095-1137
SN - 1098-660X
ER -