Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, MD 20708, USA.
Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MD 20708, USA.
Viruses. 2018 Nov 9;10(11):619. doi: 10.3390/v10110619.
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.
由于食源 RNA 病毒（如甲型肝炎病毒[HAV]）预计会出现高突变率，因此准确检测污染食品中的病毒、区分毒株和溯源仍然是一个持续存在的挑战。这导致人们预测宿主间（种间）或个体宿主内（种内）存在不止一种序列变异体。然而，尚未有关于单个受感染个体的种内变异体的报道，而且对于各种方法的单核苷酸变异（SNV）调用的准确性知之甚少。在这项研究中，通过下一代测序（NGS）和焦磷酸测序方法对核苷酸序列进行分析，确定了 HAV 临床标本之间或 HAV 克隆 1 感染的 FRhK4 细胞系列样本中病毒 SNV 的存在和身份。结果表明，种间和种内变异体在临床标本和培养样本中均同时存在。在受感染个体中发现和确认多种病毒 RNA 取决于 SNV 水平的菌株鉴别，对于成功追溯暴发源头和归因调查至关重要。对感染 FRhK4 细胞的 HAV 时间序列中 SNV 的检测提高了我们对可能由不同选择压力决定的突变动态的理解。此外，它表明 NGS 可能为其他 RNA 病毒的 SNV 检测和鉴定提供有价值的研究方法。
