Abstract Title
Hybrid Capture Genome Sequencing in Wastewater-Based Surveillance for Tracking Norovirus and Sapovirus in Alberta Communities
Presenter
Linnet Immaraj, University of Alberta
Co-Author(s)
Linnet Immaraj1, Sudha Bhavanam1, Qiang Jiang1, Judy Qiu1,2, Tiejun Gao1, Michael Parkins3, Casey Hubert4, Bonita Lee5, Xiaoli Pang1
1Department of Laboratory Medicine & Pathology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta, Canada
2Public Health Laboratory, Alberta Precision Laboratories, Edmonton, Alberta, Canada
3Departments of Medicine and Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Canada
4Department of Biological Sciences, University of Calgary, Calgary, Canada
5Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada
Abstract Category
Food & Environmental Virology-II (Wastewater & others)
Abstract
Human caliciviruses, particularly noroviruses (NoVs), cause viral gastroenteritis worldwide. Wastewater-based surveillance (WBS) using whole-genomic sequencing is a powerful tool for detecting high-risk viruses and emerging variants, supporting public health responses. This study addresses wastewater surveillance gaps by applying hybrid capture with viral probe panels (VSP) and genome sequencing, focusing on NoVs and sapoviruses (SaVs).
Viral nucleic acids were extracted from 100 mL composite wastewater samples (n = 565) collected from 12 community wastewater treatment facilities in Alberta between December 2023 and March 2025. Libraries were prepared using Illumina® RNA Prep with VSP probes targeting 200 viruses, followed by hybrid capture and sequencing on the Illumina® MiniSeq. Virus detection, coverage, and abundance were analyzed using EsViritu, where RPKMF (Reads Per Kilobase per Million Filtered reads) reflects the proportion of viral targets in each sample.
Twelve to fifteen viral targets were detected per sample. High-abundance viruses (>75% genome coverage) included polyomaviruses, gastroenteric viruses, and SARS-CoV-2, with respiratory and enteroviruses at moderate levels. Human caliciviruses were consistently detected—NoV GI (60.5%), GII (92.7%), and SaV (79.3%). Genotypes included GI.6 (dominant), GII.2, GII.4, GII.6, GII.17, along with low levels of GIV and GIX. SaV (GI.1 dominant), GII, and GV were also observed.
This study provides a snapshot of calicivirus circulation post-COVID-19, revealing diverse NoV and SaV genotypes with varying spatiotemporal abundance. Interestingly, the globally dominant NoV GII.4 exhibited low levels. These findings underscore the utility of whole-genome sequencing in WBS for real-time surveillance of NoV and SaV genotypic shifts and early detection of outbreak-prone variants.
Table- Summary of human caliciviruses detection and genotypes using comprehensive whole-genomic sequencing in wastewater
NoV detectionRPKMF abundance of NoV genotypes (Median & range)
WW sample #Positive # (%)GI GII GIV.1 and GIV.3GIX
GI GIIGI.6 Other GI genotypes GII.2 GII.4GII.6 GII.7 GII.17 Other GII genotypes
565 342 524 36.64 5.36 (0.04-85.42) 762.91 (124.07-1919.83)44.86 (6.69-232.17)41.58 (15.66- 596.07)
(60.53) (92.74) (5.13-165.61) 92.62 (30.73- 966.70)649.55 (245.46- 1855.26) 1.5 (0.05- 35.38)
0.29 (0.04- 9.06)0.11 (0.05- 0.28)
SaV detectionRPKMF abundance of SaV genotypes (Median & range)
WW sample #Positive # (%) GI GII GV.1
GIGII GI.1Other GI genotypes GII.1 Other GII genotypes
565 448 (79.29)234 (41.42)136.5 (0.02- 3693.52) 10.56 (0.02- 245.94) 8.51 (0.07- 239.06)1.35 (0.01- 636.72) 0.59(0.13-
9.83)
Viral nucleic acids were extracted from 100 mL composite wastewater samples (n = 565) collected from 12 community wastewater treatment facilities in Alberta between December 2023 and March 2025. Libraries were prepared using Illumina® RNA Prep with VSP probes targeting 200 viruses, followed by hybrid capture and sequencing on the Illumina® MiniSeq. Virus detection, coverage, and abundance were analyzed using EsViritu, where RPKMF (Reads Per Kilobase per Million Filtered reads) reflects the proportion of viral targets in each sample.
Twelve to fifteen viral targets were detected per sample. High-abundance viruses (>75% genome coverage) included polyomaviruses, gastroenteric viruses, and SARS-CoV-2, with respiratory and enteroviruses at moderate levels. Human caliciviruses were consistently detected—NoV GI (60.5%), GII (92.7%), and SaV (79.3%). Genotypes included GI.6 (dominant), GII.2, GII.4, GII.6, GII.17, along with low levels of GIV and GIX. SaV (GI.1 dominant), GII, and GV were also observed.
This study provides a snapshot of calicivirus circulation post-COVID-19, revealing diverse NoV and SaV genotypes with varying spatiotemporal abundance. Interestingly, the globally dominant NoV GII.4 exhibited low levels. These findings underscore the utility of whole-genome sequencing in WBS for real-time surveillance of NoV and SaV genotypic shifts and early detection of outbreak-prone variants.
Table- Summary of human caliciviruses detection and genotypes using comprehensive whole-genomic sequencing in wastewater
NoV detectionRPKMF abundance of NoV genotypes (Median & range)
WW sample #Positive # (%)GI GII GIV.1 and GIV.3GIX
GI GIIGI.6 Other GI genotypes GII.2 GII.4GII.6 GII.7 GII.17 Other GII genotypes
565 342 524 36.64 5.36 (0.04-85.42) 762.91 (124.07-1919.83)44.86 (6.69-232.17)41.58 (15.66- 596.07)
(60.53) (92.74) (5.13-165.61) 92.62 (30.73- 966.70)649.55 (245.46- 1855.26) 1.5 (0.05- 35.38)
0.29 (0.04- 9.06)0.11 (0.05- 0.28)
SaV detectionRPKMF abundance of SaV genotypes (Median & range)
WW sample #Positive # (%) GI GII GV.1
GIGII GI.1Other GI genotypes GII.1 Other GII genotypes
565 448 (79.29)234 (41.42)136.5 (0.02- 3693.52) 10.56 (0.02- 245.94) 8.51 (0.07- 239.06)1.35 (0.01- 636.72) 0.59(0.13-
9.83)