Development of a direct whole genome sequencing for hepatitis A virus from serum and analysis of genetic characteristics

Abstract

Abstract Hepatitis A virus (HAV) is transmitted via the fecal–oral route, including through person-to-person contact and consumption of contaminated food. In 2019, 17,638 cases of HAV infection were reported in South Korea. The use of whole-genome sequencing (WGS) for rapid and accurate epidemiological investigation of HAV remains challenging due to the low viral titers and the lack of a cell culture system. Here, we developed a multiplex PCR (mPCR)-based next generation sequencing (NGS) method for direct WGS of HAV from serum using the Illumina platform. Overlapping primers were designed to generate amplicons covering the entire HAV genome, enabling successful sequencing from samples with viral titers as low as 3.0 log10 copies/µL. The method achieved a mapping rate of 98.95% and 98.32% genome coverage. All nine HAV strains analyzed were classified as genotype IA and showed > 99% sequence homology. Phylogenetic analysis of the whole genome, structural, and non-structural regions demonstrated clustering with HAV strains previously isolated in Japan. Correlation coefficients between WGS and individual gene regions approached 1.0, confirming the method’s reliability for molecular epidemiology. This mPCR-based NGS approach provides a robust tool for HAV surveillance and facilitates high-resolution genomic analysis from non-culturable clinical samples.

Keywords

• Hepatitis A virus
• Illumina platform
• Whole-genome sequencing
• Phylogeny
• Dendrogram analysis