Nottingham Digestive Diseases Centre, School of Medicine, University of Nottingham, UK; NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and the University of Nottingham, UK; MRC/EPSRC Nottingham Molecular Pathology Node, University of Nottingham, UK.
Instituto Adolfo Lutz, Brazilian Ministry of Health, São Paulo, Brazil.
J Clin Virol. 2020 Aug;129:104483. doi: 10.1016/j.jcv.2020.104483. Epub 2020 Jun 2.
Effective drug regimens for the treatment of hepatitis B virus (HBV) infections are essential to achieve the World Health Organisation commitment to eliminate viral hepatitis by 2030. Lamivudine (3TC) is widely used in countries with high levels of chronic HBV, however resistance has been shown to occur in up to 50 % of individuals receiving continuous monotherapy for 4 years. Telbivudine (LdT) is now more commonly used in place of lamivudine but is ineffective against 3TC-resistant HBV. Genotyping and identification of resistanceassociated substitutions (RAS) is not practical in many locations.
A novel assay was designed to enable HBV genotyping and characterisation of resistance mutations directly from serum samples stored on filter paper, using Sanger and MinION sequencing.
The assay was applied to a cohort of 30 samples stored on filter paper for several years with HBV viral loads ranging from 8.2 × 10 to 635 IU/mL. A set of 6 high-titre samples were used in a proof-of-principle study using the MinION sequencer.
The assay allowed determination of HBV genotype and elucidation of RAS down to 600 IU/mL using a 550bp amplicon. Sequencing of a 1.2 kb amplicon using a MinION sequencer gave results consistent with Sanger sequencing and allowed the identification of minor populations of variants.
We present two approaches for reliable HBV sequencing and RAS identification using methods suitable for resource-limited environments. This is the first demonstration of extraction-free DNA sequencing direct from DSS using MinION and these workflows are adaptable to the investigation of other DNA viruses.
有效的乙型肝炎病毒 (HBV) 感染治疗方案对于实现世界卫生组织到 2030 年消除病毒性肝炎的承诺至关重要。拉米夫定（3TC）在慢性 HBV 水平较高的国家被广泛使用，但已显示出在接受连续单药治疗 4 年的个体中，多达 50%的个体出现耐药。替比夫定（LdT）现在更常用于替代拉米夫定，但对 3TC 耐药的 HBV 无效。在许多地方，基因分型和耐药相关取代（RAS）的鉴定并不实用。
设计了一种新的检测方法，可直接从储存在滤纸上的血清样本中进行 HBV 基因分型和耐药突变鉴定，使用 Sanger 和 MinION 测序。
该检测方法应用于一组在滤纸上储存多年的 30 个样本，HBV 病毒载量范围为 8.2×10 至 635IU/mL。一组 6 个高病毒载量样本用于使用 MinION 测序仪进行原理验证研究。
该检测方法允许使用 550bp 扩增子确定 HBV 基因型并阐明耐药突变，最低检测限为 600IU/mL。使用 MinION 测序仪对 1.2kb 扩增子进行测序可得到与 Sanger 测序一致的结果，并可鉴定出少量变异体。
我们提出了两种使用适合资源有限环境的方法进行可靠的 HBV 测序和 RAS 鉴定的方法。这是首次直接从 DSS 使用 MinION 进行无提取 DNA 测序的演示，这些工作流程可适用于其他 DNA 病毒的研究。
