Long Amplicon Nanopore Sequencing for Dual-Typing RdRp and VP1 Genes of Norovirus Genogroups I and II in Wastewater

IF 4.1 2区 农林科学 Q2 ENVIRONMENTAL SCIENCES Food and Environmental Virology Pub Date : 2024-09-06 DOI:10.1007/s12560-024-09611-5
G. Scott, D. Ryder, M. Buckley, R. Hill, S. Treagus, T. Stapleton, D. I. Walker, J. Lowther, F. M. Batista
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Abstract

Noroviruses (NoVs) are the leading cause of non-bacterial gastroenteritis with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve outbreak tracking and source apportionment. Here, we have developed such a method with a consensus-based bioinformatics pipeline and optimised reverse transcription (RT) and PCR procedures. Inhibitor removal and LunaScript® RT gave robust amplification of the ≈ 1000 bp RdRP + VP1 amplicon for both the GI and GII PCR assays. Platinum™ Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3 and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also frequently detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population.

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用于对废水中诺如病毒 I 和 II 基因组的 RdRp 和 VP1 基因进行双重定型的长扩增片段纳米孔测序。
诺罗病毒(NoVs)是非细菌性肠胃炎的主要病因,每年造成的社会成本高达 603 亿美元。开发一种基于长扩增片段纳米孔的方法,从单个 RNA 片段对 RNA 依赖性 RNA 聚合酶(RdRp)和主要结构蛋白(VP1)区域进行双重分型,可以改进现有的诺罗病毒分型方法。将其应用于基于废水的流行病学(WBE)和环境检测可发现新型病毒,并改进疫情追踪和病毒源划分。在这里,我们利用基于共识的生物信息学管道和优化的反转录(RT)和 PCR 程序开发了这种方法。在 GI 和 GII PCR 检测中,去除抑制剂和 LunaScript® RT 能强力扩增 ≈ 1000 bp RdRP + VP1 扩增片段。与其他聚合酶相比,Platinum™ Taq 聚合酶显示出良好的灵敏度,并降低了非特异性扩增 (NSA) 的水平。优化的 PCR 退火温度大大降低了非特异性扩增(GI 和 GII 分别为 51.3% 和 42.4%),提高了产量(GII 为 86.5%),并增加了 GII 的分类群丰富度(57.7%)。对三份 NoV 阳性粪便样本的分析表明,桑格测序的核苷酸相似度为 100%。在废水中检测到 8 种 GI 基因型、11 种聚合酶类型(p 型)和 13 种组合,以及 4 种 GII 基因型、4 种 p 型和 8 种组合;这突显了英格兰废水中诺如病毒类群的多样性。在临床样本中检测到的最常见基因型在废水中也全部检测到,同时我们还经常检测到临床数据中未报告的几种 GI 基因型。因此,将这种方法应用到 WBE 计划中可以更准确地测量人口中诺如病毒的多样性。
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来源期刊
Food and Environmental Virology
Food and Environmental Virology ENVIRONMENTAL SCIENCES-MICROBIOLOGY
CiteScore
6.50
自引率
2.90%
发文量
35
审稿时长
1 months
期刊介绍: Food and Environmental Virology publishes original articles, notes and review articles on any aspect relating to the transmission of pathogenic viruses via the environment (water, air, soil etc.) and foods. This includes epidemiological studies, identification of novel or emerging pathogens, methods of analysis or characterisation, studies on survival and elimination, and development of procedural controls for industrial processes, e.g. HACCP plans. The journal will cover all aspects of this important area, and encompass studies on any human, animal, and plant pathogenic virus which is capable of transmission via the environment or food.
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