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

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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by Agencia Nacional de Promocion de la Investigacion, el Desarrollo Tecnologico y la Innovacion de Argentina [grant number PICT-2021-00041]. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was conducted according to the guidelines of the Declaration of Helsinki (1964, amended most recently in 2008) of the World Medical Association, and in accordance with specific local ethical regulations (Legislature of the Province of Cordoba, 2009). The Research Evaluation Committee of the Institute of Virology Dr. Jose Maria Vanella (National University of Cordoba) evaluated the ethics of this study and approved its conduct. Sample IDs were not known to anyone outside the research group. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.