Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

Abstract

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

Near-Complete Genome Sequence of a Human Norovirus GII.1[Pg] Strain Associated with Acute Gastroenteritis, Determined Using Long-Read Sequencing

“…Human noroviruses cluster in genogroups I, II, and IV ( 2 , 3 ). Due to the high genetic diversity of noroviruses, in addition to PCR-based methods and partial sequencing approaches, high-throughput whole-genome sequencing, which provides nucleotide information on the complete genome, is needed for accurate characterization of these viruses ( 4 , 5 ). In comparison with short-read sequencing, Nanopore long-read sequencing technologies make it possible to detect and identify viruses faster as well as accurately characterize the genomic complexity with complete coverage of the entire genome by a single read ( 6 ).…”

Hepatitis A infections from food

“…The very novel analytical methods applied to detect and characterize HAV in food and environmental samples rely on next generation sequencing (NGS) techniques. The wide potential of NGS includes the characterization of viral quasispecies heterogeneity (Yang et al 2018) and the metagenomics approach represent a promising solution for detecting all viruses (virome), delivering genomic information and discovering unknown viruses (Nieuwenhuijse and Koopmans 2017;Yang et al 2017). NGS have been successfully applied for amplification-independent detection of norovirus and HAV spiked in celery samples at low copy using both targeted and metagenomics approaches (Yang et al 2017).…”

“…The wide potential of NGS includes the characterization of viral quasispecies heterogeneity (Yang et al . 2018) and the metagenomics approach represent a promising solution for detecting all viruses (virome), delivering genomic information and discovering unknown viruses (Nieuwenhuijse and Koopmans 2017; Yang et al . 2017).…”

Rescuing eGFP-Tagged Canine Distemper Virus for 40 Serial Passages Separately in Ribavirin- and Non-Treated Cells: Comparative Analysis of Viral Mutation Profiles

“…As an alternative technique, the NGS has been widely applied to estimate the exceptionally-high diversity within viral quasispecies. By means of it, single-nucleotide polymorphisms (SNPs) in various viruses can be systematically analyzed for revealing their evolutionary dynamics ( Campo et al., 2014 ; Hasing et al., 2016 ; Ni et al., 2016 ; Yang et al., 2018 ; Liu et al., 2021 ).…”