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

Zhihui Yang,Michael Kulka,Chris A Whitehouse,Diana Ngo,Mark Mammel

doi:10.3390/v10110619

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.

Highlights

The majority of the known foodborne viruses, either linked or found directly responsible for foodborne illness, have RNA genomes [1]
We concluded in our previous study that both read coverage and nucleotide frequency at a given position are significant for the single nucleotide variations (SNVs) calling from next-generation sequencing (NGS) data and pyrosequencing confirmation [24]
In determining the sequence variation of SNVs and small indels, it usually requires an average depth of 15× and 33× to detect homozygous SNVs and the same proportion of heterozygous SNVs, respectively, based on the studies on human genome sequencing with the Illumina platform [35,36]

Summary

Introduction

The majority of the known foodborne viruses, either linked or found directly responsible for foodborne illness, have RNA genomes [1]. RNA viruses have been shown to exhibit high mutation rates primarily due to the low-fidelity of RNA polymerases [2] and absence of post-replication nucleotide repair mechanisms [3,4]. These RNA viruses are generally expected to exist as populations of non-identical but closely genetic-related viral variants between the hosts (inter-host) or within an individual host (intra-host), which are referred to as quasispecies [5,6]. Viral genetic heterogeneity, generated by single nucleotide variations (SNVs), is believed to be a strategy of virus evolution and virus adaptability [4]. The presence of more than one sequence variant is a challenge for accurate virus detection, identification, and source attribution of contaminated food items.

Methods

Results

Conclusion