Department of Preservation and Food Safety Technologies, IATA-CSIC, Valencia, Spain.
ADM-Lifesequencing - Health and Wellness - Adm Nutrition, Valencia, Spain.
J Sci Food Agric. 2023 Jul;103(9):4450-4457. doi: 10.1002/jsfa.12522. Epub 2023 Mar 7.
The virome (i.e. community of mainly RNA and DNA eukaryotic viruses and bacteriophages) of waters is yet to be extensively explored. In particular, the virome of waters used for irrigation could therefore potentially carry viral pathogens that can contaminate fresh produce. One problem in obtaining viral sequences from irrigation waters is the relatively low amount of virus particles, as well as the presence of human, bacterial and protozoan cells. The present aimed study was to compare different processing, amplification, and sequencing approaches for virome characterization in irrigation waters.
Our analyses considered percentages of viral reads, values for diversity indices and number of families found in sequencing results. The results obtained suggest that enrichment protocols using two (bezonase and microccocal nuclease) or four enzymes at once (bezonase, microccocal nuclease, DNAse and RNase), regardless of an Amicon filtration step, are more appropriate than separated enzymatic treatments for virome characterization in irrigation water. The NetoVIR protocol combined with the ScriptSeq v2 RNA-Seq Library (P0-L20 protocol) showed the highest percentages of RNA viruses and identified the higher number of families.
Although virome characterization applied in irrigation waters is an important tool for protecting public health by informing on circulating human and zoonotic infections, optimized and standardized procedures should be followed to reduce the variability of results related to either the sample itself and the downstream bioinformatics analyses. Our results show that virome characterization can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided that appropriate and rigorous controls are included. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
水体中的病毒组（即主要由 RNA 和 DNA 真核病毒和噬菌体组成的群落）尚未得到广泛探索。特别是，用于灌溉的水体中的病毒组可能携带可污染新鲜农产品的病毒病原体。从灌溉水中获取病毒序列的一个问题是病毒颗粒的相对数量较少，以及存在人类、细菌和原生动物细胞。本研究旨在比较不同的处理、扩增和测序方法，以用于灌溉水中的病毒组特征描述。
我们的分析考虑了病毒读数的百分比、多样性指数的值以及测序结果中发现的家族数量。结果表明，使用两种（贝诺酶和微球菌核酸酶）或同时使用四种酶（贝诺酶、微球菌核酸酶、DNA 酶和 RNA 酶）的富集方案，无论是否进行 Amicon 过滤步骤，都比单独使用酶处理更适合用于灌溉水中的病毒组特征描述。NetoVIR 方案与 ScriptSeq v2 RNA-Seq 文库（P0-L20 方案）结合使用，显示出更高比例的 RNA 病毒，并鉴定出更高数量的家族。
尽管应用于灌溉水的病毒组特征描述是通过告知循环中的人类和人畜共患病感染来保护公众健康的重要工具，但应遵循优化和标准化的程序，以减少与样品本身和下游生物信息学分析相关的结果变异性。我们的结果表明，病毒组特征描述可以成为发现环境中致病病毒的重要工具，并可用于告知和优化基于参考的检测方法，前提是包含适当和严格的对照。
