Molecular diagnosis of patients with hepatitis A virus infection using amplicon-based nanopore sequencing

Abstract

High-throughput sequencing is a robust tool used for identifying and tracking pathogen outbreaks. Whole-genome sequencing of hepatitis A virus (HAV) remains poor due to ultra-low viral loads, limitations of next-generation sequencing technology, and its high costs in clinical applications. This study evaluated multiplex polymerase chain reaction (PCR)-based nanopore sequencing to obtain whole-genome sequences of HAV. The HAV genomes were obtained directly from patient specimens for a rapid molecular diagnosis of viral genotypes. Serum and stool samples were collected from six patients with hepatitis A infection. Amplicon- based nanopore sequencing was performed from the clinical specimens to identify HAV genotypes by acquiring nearly complete-genome sequences. TaqMan-based quantitative PCR (qPCR) was conducted to detect and quantify multiple HAV genes. Singleplex-based nanopore sequencing demonstrated high genome coverage rates (90.4-99.5%) of HAV within 8 h, at viral RNA loads of 10 to 105 copies/μL. TaqMan qPCR showed multiplex quantification of HAV genes namely, VP0, VP3, and 3C. This study provides useful insights into rapid molecular diagnosis during hepatitis A outbreaks and may ultimately augment public health disease surveillance in the hospital and epidemiology field.