1 Inter-and intra-host nucleotide variations in hepatitis 2 A virus in culture and clinical samples detected by 3 next-generation sequencing 4

The accurate virus detection, strain discrimination, and source attribution of contaminated 12 food items remains a persistent challenge because of the high mutation rates anticipated to occur in 13 foodborne RNA viruses, such as Hepatitis A virus (HAV). This has led to predictions of the existence 14 of more than one sequence variant between the hosts (inter-host) or within an individual host (intra15 host). However, there have been no reports of intra-host variants from an infected single individual, 16 and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various 17 methods. In this study, the presence and identity of viral SNVs, either between HAV clinical 18 specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were 19 determined following analyses of nucleotide sequences generated using next-generation 20 sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter21 and intra-host variants both in the clinical specimens and the cultured samples. The discovery and 22 confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination 23 at the SNV level, and critical for successful outbreak traceback and source attribution investigations. 24 The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding 25 on the mutation dynamics determined probably by different selective pressures. Additionally, it 26 demonstrated that NGS could potentially provide a valuable investigative approach toward SNV 27 detection and identification for other RNA viruses. 28