Novel strategy for whole-genome sequencing of hepatitis A virus using NGS illumina technology and phylogenetic comparison with partial VP1/2A genomic region.

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. We developed an amplicon-based next-generation sequencing (NGS) strategy to obtain complete HAV genomes utilizing the COVIDSeq Test (Illumina). Twenty-five primer pairs were designed and used to amplify partial genomic fragments (400 bp) that comprise the entire HAV genome sequence from previously HAV 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. Eleven samples were amplified and sequenced, with coverage between 79.3 and 100% (> 90% in 9 samples). Although phylogenetic analyses of partial sequences allowed genotype and subgenotype identification, WGS analyses yielded more accurate and reliable results for the phylogeny (phylogenetic definition). The amplicon-based NGS WGS tool developed by adapting the COVIDSeq test to HAV proved to be efficient. The study of partial VP1/2A regions (mainly the 1084 bp fragment) would constitute useful alternatives for outbreak investigation and surveillance when WGS could not be performed.