Amplicon-Based High-Throughput Sequencing Method for Genotypic Characterization of Norovirus in Oysters

Fitzpatrick, Amy; Rupnik, Agnieszka; O’Shea, Helen; Crispie, Fiona; Cotter, Paul D.; Keaveney, Sinéad

doi:10.1128/aem.02165-22

Cited by 2 publications

(2 citation statements)

References 76 publications

(102 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, increased multiplexing as well as additional optimization to increase target read proportions could lower per-sample costs. Furthermore, we anticipate the value of this approach to be the greatest for supporting food-borne surveillance, where PCR-based amplification is limited by low, degraded viral input and the presence of inhibitors ( 61 , 62 ). For food specimen in particular, optimization should evaluate the effect of pH and organic load, as well as nucleic acid shearing during virus extraction, on enrichment.…”

Section: Discussionmentioning

confidence: 99%

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

Zufan,

Mercoulia,

Kwong

et al. 2024

Microbiol Spectr

View full text Add to dashboard Cite

Hepatitis A virus (HAV) infections are an increasing public health concern in low-endemicity regions due to outbreaks from food-borne infections and sustained transmission among vulnerable groups, including persons experiencing homelessness, those who inject drugs, and men who have sex with men, which is further compounded by aging, unvaccinated populations. DNA sequence characterization of HAV for source tracking is performed by comparing small subgenomic regions of the virus. While this approach has been successful when robust epidemiological data are available, poor genetic resolution can lead to the conflation of outbreaks with sporadic cases. HAV outbreak investigations would greatly benefit from the additional phylogenetic resolution obtained by whole virus genome sequence comparisons. However, HAV genomic approaches can be difficult because of challenges in isolating the virus, low sensitivity of direct metagenomic sequencing in complex sample matrices like various foods, such as fruits, vegetables, and molluscs, and difficulty designing highly multiplexed PCR primers across diverse HAV genotypes. Here, we introduce a proof-of-concept pan-HAV oligonucleotide hybrid capture enrichment assay from serum and frozen berry specimens that yields complete and near-complete HAV genomes from as few as four input HAV genome copies. We used this method to recover HAV genomes from human serum specimens with high Cτ values (34.7–42.7), with high assay performance for all six human HAV subgenotypes, both contemporary and historical. Our approach provides a highly sensitive and streamlined workflow for HAV whole-genome sequencing from diverse sample types that can be the basis for harmonized and high-resolution molecular epidemiology during HAV outbreak surveillance. IMPORTANCE This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging sample matrices can be accommodated, making them suitable for broad implementation in clinical and public health laboratories. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations as well as our understanding of the global diversity and transmission dynamics of HAV.

show abstract

Section: Discussionmentioning

confidence: 99%

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

Zufan,

Mercoulia,

Kwong

et al. 2024

Microbiol Spectr

View full text Add to dashboard Cite

show abstract

“…Furthermore, we anticipate the value of this approach to be the greatest for supporting food-borne surveillance, where PCR-based amplification is limited by low, degraded viral input and the presence of inhibitors. 60,61 For food specimen in particular, optimisation should evaluate the effect of pH and organic load, as well as nucleic acid shearing during virus extraction, on enrichment. Finally, overnight hybridisation was used to accommodate hands-on staffing schedules.…”

Section: Discussionmentioning

confidence: 99%

High performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

Zufan

Mercoulia

Kwong

et al. 2023

Preprint

View full text Add to dashboard Cite

Hepatitis A virus (HAV) infections are an increasing public health concern in low-endemicity regions due to outbreaks from foodborne infections and sustained transmission among vulnerable groups, including persons experiencing homelessness, those who inject drugs, and men who have sex with men (MSM), which is further compounded by aging, unvaccinated populations. DNA sequence characterisation of HAV for source tracking is performed by comparing small subgenomic regions of the virus. While this approach has been successful when robust epidemiological data are available, poor genetic resolution can lead to conflation of outbreaks with sporadic cases. HAV outbreak investigations would greatly benefit from the additional phylogenetic resolution obtained by whole virus genome sequence comparisons. However, HAV genomic approaches can be difficult because of challenges in isolating the virus, low sensitivity of direct metagenomic sequencing in complex sample matrices like various foods such as fruits, vegetables and molluscs, and difficulty designing highly multiplexed PCR primers across diverse HAV genotypes. Here, we introduce a proof-of-concept pan-HAV oligonucleotide hybrid capture enrichment assay from serum and frozen berry specimens that yields complete and near-complete HAV genomes from as few as four input HAV genome copies. We used this method to recover HAV genomes from human serum specimens with high CT values (34.7-42.7), with high assay performance for all six human HAV sub-genotypes, both contemporary and historical. Our approach provides a highly sensitive and streamlined workflow for HAV WGS from diverse sample types, that can be the basis for harmonised and high-resolution molecular epidemiology during HAV outbreak surveillance.

show abstract

Amplicon-Based High-Throughput Sequencing Method for Genotypic Characterization of Norovirus in Oysters

Abstract: While foodborne outbreaks account for approximately 14% of norovirus cases (L. Verhoef, J.

Cited by 2 publications

References 76 publications

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

High performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

Contact Info

Product

Resources

About