MRC-University of Glasgow Centre for Virus Research, Glasgow, UK.
Blood Borne Virus Unit, Virus Reference Department, National Infection Service, Public Health England, London, UK.
J Clin Virol. 2021 Jun;139:104738. doi: 10.1016/j.jcv.2021.104738. Epub 2021 Jan 27.
Hepatitis E Virus (HEV) is emerging as a public health concern across Europe and tools for complete genome data to aid epidemiological and virulence analysis are needed. The high sequence heterogeneity observed amongst HEV genotypes has restricted most analyses to subgenomic regions using PCR-based methods, which can be unreliable due to poor primer homology. We designed a panel of custom-designed RNA probes complementary to all published HEV full genome NCBI sequences. A target enrichment protocol was performed according to the NimbleGen® standard protocol for Illumina® library preparation. Optimisation of this protocol was performed using 40 HEV RNA-positive serum samples and the World Health Organization International Reference Panel for Hepatitis E Virus RNA Genotypes for Nucleic Acid Amplification Technique (NAT)-Based Assays and related reference materials. Deep sequencing using this target enrichment protocol resulted in whole genome consensus sequences from samples with a viral load range of 1.25 × 10-1.17 × 10 IU/mL. Phylogenetic analysis of these sequences recapitulated and extended the partial genome results obtained from genotyping by Sanger sequencing (genotype 1, ten samples and genotype 3, 30 samples). The protocol is highly adaptable to automation and could be used to sequence full genomes of large sample numbers. A more comprehensive understanding of hepatitis E virus transmission, epidemiology and viral phenotype prediction supported by an efficient method of sequencing the whole viral genome will facilitate public health initiatives to reduce the prevalence and mitigate the harm of HEV infection in Europe.
戊型肝炎病毒（HEV）在欧洲各地正成为一个公共卫生关注点，因此需要能够提供完整基因组数据以辅助流行病学和毒力分析的工具。由于引物同源性差，HEV 基因型之间观察到的高序列异质性限制了大多数基于 PCR 的亚基因组区域分析。我们设计了一组与所有已发表的 HEV 全基因组 NCBI 序列互补的定制 RNA 探针。根据 NimbleGen®针对 Illumina®文库制备的标准协议进行了靶向富集方案。使用 40 份 HEV RNA 阳性血清样本和世界卫生组织（WHO）用于核酸扩增技术（NAT）的丙型肝炎病毒 RNA 基因分型国际参考面板（NAT）以及相关参考材料，对该方案进行了优化。使用此靶向富集方案进行深度测序，可从病毒载量范围为 1.25×10-1.17×10 IU/mL 的样本中获得全基因组一致序列。对这些序列的系统发育分析再现并扩展了通过 Sanger 测序（基因型 1，10 个样本和基因型 3，30 个样本）进行基因分型获得的部分基因组结果。该方案具有高度的自动化适应性，可用于对大量样本进行全基因组测序。通过高效的全病毒基因组测序方法，对戊型肝炎病毒传播、流行病学和病毒表型预测有更全面的了解，将有助于欧洲减少 HEV 感染的流行和减轻其危害的公共卫生措施。
