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

ABSTRACT 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.

speculate about the application to environmental samples?What normally happens with a sample of a CT value of 38 or >40?Is this normally seen as positive?
Is there a reference for use of the LENTICULE discs?Since GenBank contains many partial sequences of HAV, is it of use to note the parameters used to download the chosen sequences from GenBank? Line 289 should be cost-effective approach.
In lines 291-292 the differences in berry and serum samples are discussed.What is the impact of pH and organic load?Was the pH and other characteristic measured as they would be important in further enhancing the use of this tool.
The raw data in the supplemental files is likely to be useful to some researchers.
Reviewer #2 (Comments for the Author): The authors developed an oligonucleotide hybrid capture enrichment-based sequencing method to detect the hepatitis A virus.They demonstrated this method is highly sensitive and could be promising for future hepatitis A virus outbreak surveillance.
There are some problems that should be addressed.
Only phylogeny was carried out and genomic analysis was not sufficient for the comparison between the near-complete genomes and their reference genomes to see how different or similar they were.The authors should perform more genomic analysis for viruses (e.g.genome annotation) to look into the data at a deeper level.It is more important to see how the nearcomplete genomes performed in downstream genomic analysis commonly performed for viral genomes.Any reason why different sequencing platforms (iSeq and NextSeq) were used for the optimization experiment and clinical samples?It would be helpful if you could provide a flow chart to display your workflow, including experiments and subsequent bioinformatic analysis.Did you test the difference between using SPAdes and metaSPAdes for the filtered reads?Lines 117 and 119: used Lines 137 and 139: 222 μL...200 μL.A space should be added before μL throughout the manuscript.

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