Division of Infectious Disease Diagnostics, National Institute for Biological Standards and Control (NIBSC), South Mimms EN6 3QG, UK.
Division of Analytical and Biological Sciences, National Institute for Biological Standards and Control (NIBSC), Medicines and Healthcare Product Regulatory Agency (MHRA), South Mimms EN6 3QG, UK.
Viruses. 2023 May 30;15(6):1289. doi: 10.3390/v15061289.
Reactivation of JC and BK polyomaviruses during immunosuppression can lead to adverse clinical outcomes. In renal transplant recipients, BKV-associated nephropathy can result in graft loss, while in patients with autoimmune disorders, prolonged immunomodulatory drug use can cause rare onset of progressive multifocal leukoencephalopathy due to JCV reactivation. In such patients, accurate BK and JC viral load determinations by molecular technologies are important for diagnosis and clinical management; however, comparability across centres requires effective standardisation of diagnostic molecular detection systems. In October 2015, the WHO Expert Committee for Biological Standardisation (ECBS) established the 1st WHO International Standards (ISs) for use as primary-order calibrants for BKV and JCV nucleic acid detection. Two multi-centre collaborative studies confirmed their utility in harmonising agreement across the wide range of BKV and JCV assays, respectively. Previous Illumina-based deep sequence analysis of these standards, however, identified deletions in different regions, including the large T-antigen coding region. Hence, further detailed characterization was warranted.
Comprehensive sequence characterisation of each preparation using short- and long-read next-generation sequencing technologies was performed with additional corroborative independent digital PCR (dPCR) determinations. Potential error rates associated with long-read sequencing were minimised by applying rolling circle amplification (RCA) protocols for viral DNA (circular dsDNA), generating a full validation of sequence identity and composition and delineating the integrity of full-length BK and JC genomes.
The analysed genomes displayed subpopulations frequently characterised by complex gene re-arrangements, duplications and deletions.
Despite the recognition of such polymorphisms using high-resolution sequencing methodologies, the ability of these reference materials to act to enhance assay harmonisation did not appear significantly impacted, based on data generated by the 2015 WHO collaborative studies, but highlights cautionary aspects of IS generation and commutability for clinical molecular diagnostic application.
免疫抑制期间 JC 和 BK 多瘤病毒的再激活可导致不良的临床结局。在肾移植受者中，BKV 相关性肾病可导致移植物丢失，而在自身免疫性疾病患者中，由于 JCV 再激活，长期使用免疫调节药物可导致罕见的进行性多灶性白质脑病的发生。在这些患者中，通过分子技术准确测定 BK 和 JC 病毒载量对于诊断和临床管理非常重要；然而，中心间的可比性需要对诊断性分子检测系统进行有效的标准化。2015 年 10 月，世界卫生组织生物标准化专家委员会（ECBS）建立了第 1 批世界卫生组织国际标准（IS），用作 BKV 和 JCV 核酸检测的一级校准标准。两项多中心合作研究分别证实了它们在协调广泛的 BKV 和 JCV 检测之间的一致性方面的效用。然而，以前对这些标准进行的基于 Illumina 的深度测序分析发现，它们在不同区域（包括大 T 抗原编码区）存在缺失。因此，有必要进行进一步的详细特征描述。
使用短读长和长读长下一代测序技术对每个制剂进行全面的序列特征描述，并进行额外的独立数字 PCR（dPCR）确证。通过应用滚环扩增（RCA）方案对病毒 DNA（环状 dsDNA）进行长读长测序，可将潜在的错误率最小化，从而全面验证序列同一性和组成，并描绘 BK 和 JC 全长基因组的完整性。
分析的基因组显示亚群经常具有复杂的基因重排、重复和缺失。
尽管使用高分辨率测序方法识别了这些多态性，但这些参考物质增强检测一致性的能力似乎并未受到显著影响，这是基于 2015 年世界卫生组织合作研究生成的数据，但突出了 IS 生成和临床分子诊断应用的可互换性的谨慎方面。
