Available online 12 November 2022, 114649

A custom AmpliSeq Panel assay generated hepatitis A whole-genome sequences.

The assay can generate 16 hepatitis A whole genomes in 24 hours on a single run.

Whole-genome sequencing further distinguishes closely related sequences.

The assay identified a potentially significant mutation in the IRES element.

Whole-genome sequencing is useful for genotyping and outbreaks analysis.

Since 2016, the United States has experienced a resurgence in the number of hepatitis A virus (HAV) cases and outbreaks. These outbreaks have been sustained by person-to-person transmission with cases occurring predominantly in high-risk populations including intravenous drug users, individuals experiencing homelessness, and men who have sex with men. To investigate HAV transmission, a molecular-surveillance system consisting of real-time RT-PCR (rRT-PCR) for detection, and a conventional RT-PCR assay for genotyping of HAV, was established in New York State (NYS) in 2019. Since then, a total of 271 HAV-positive serum samples collected from cases across NYS between 2019 and 2021 were identified by rRT-PCR. To rapidly and efficiently generate HAV whole-genome sequences, a custom AmpliSeqTM panel was designed in collaboration with Thermo Fisher. To streamline the process, sample preparation was performed on an Ion Chef and sequencing on an Ion S5XL. Of the 271 HAV-positive samples, the whole-genome sequencing (WGS) assay successfully generated 134 near-complete, high-quality HAV sequences. Phylogenetic analysis of the VP1-2A region identified 216 IB, 48 IA, and 2 IIIA genotypes, while 5 were unable to be typed due to poor sequence in this key region. The HAV whole-genome sequencing approach provided a more efficient and streamlined approach for genotyping HAV compared to previous methods and resulted in phylogenetic trees with enhanced resolution compared to the HAV VP1-2A region alone.

Hepatitis A

AmpliSeqTM

whole-genome sequencing

GHOST

molecular epidemiology

© 2022 Published by Elsevier B.V.