Hepatitis A virus whole genome sequencing strategy using NGS/Illumina technology

Abstract

Background: Molecular epidemiology of hepatitis A virus (HAV) plays a critical role in identifying outbreak origin and conducting surveillance. Although it is mostly carried out using short partial VP1/2A genomic sequences, utilizing whole-genome sequences (WGS) provides more accurate and robust information. Objective: we aimed to develop an amplicon-based next-generation sequencing (NGS) strategy to obtain complete HAV genomes utilizing the COVIDSeq Test (Illumina), available in surveillance laboratories after the COVID19 pandemic. Study design: 25 primer pairs were designed and used to amplify partial genomic fragments (400bp) that comprise the entire HAV genome sequence from 16 HAV previously positive serum and stool samples from Argentina. The DNA library was prepared using the Illumina COVIDSeq Test and sequenced in a MiSeq equipment. Phylogenetic analyses were performed with IQ-Tree using WGS and VP1/2A partial sequences of 1084pb and 422pb. Results: 11 samples could be successfully amplified and sequenced, with horizontal coverage between 79.3%-100% (>90% in 9 samples). Individual RT-PCRs and Sanger sequencing with specific primers had to be performed in 6 samples to cover gaps of Ns. Phylogenetic analyses of WGS and VP1/2A partial sequences yielded similar results (clustering with genotype IA), although 422pb fragments showed low accurate grouping definition. Conclusion: the amplicon-based NGS whole genome sequencing tool developed by adapting the COVIDSeq test to HAV, proved to be efficient in generating new complete and near-complete viral sequences. The study of the 1084bp fragment of the VP1/2A region would constitute a useful alternative option for outbreak investigation with public health impact.