Departments of Biochemistry and Molecular Genetics, Israel Institute for Biological Research (IIBR), Ness Ziona 74100, Israel.
Clinical Microbiology, Sheba Medical Center, Ramat-Gan 52621, Israel.
Viruses. 2022 Aug 18;14(8):1817. doi: 10.3390/v14081817.
As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and the population's immunity to other OPV-related diseases such as MPX. In parallel, there was a need for differential diagnosis between the different OPVs' clinical manifestations and diseases with similar symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a rapid genetic-based diagnostic tool for accurate and specific identification of MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic regions that harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5'-phosphonate were simultaneously added, thus making the PCR product ligation ready for nanopore sequencing. Following rapid sequencing (a few minutes), the reads were compared to a reference database and the nearest strain was identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced during the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we obtained definite results, identifying other vesicle-forming viruses: Human herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was a proof-of-concept study, demonstrating the potential of the RANS approach for rapid and discriminatory identification of a panel of closely related pathogens. The simplicity and affordability of our approach makes it straightforward to implement in any genetics lab. Moreover, other differential diagnostics panels might benefit from the implementation of the RANS approach into their diagnostics pipelines.
截至 2022 年 7 月，全球已报告超过 16000 例实验室确诊的猴痘（MPX）病例。直到最近，MPX 还是一种罕见的病毒性疾病，在非洲以外很少被发现。猴痘病毒（MPXV）属于正痘病毒（OPV）属，是天花病毒（引起天花的病原体）的遗传近亲。天花根除后，与天花相关的发病率显著下降，人群对其他正痘病毒相关疾病（如猴痘）的免疫力也下降。同时，需要对不同 OPV 的临床表现和具有相似症状的疾病（如水痘、单纯疱疹）进行鉴别诊断。本研究旨在提供一种快速的基于遗传的诊断工具，用于准确和特异性地识别 MPXV 和其他相关水疱形成病原体。我们最初汇编了一份包含 14 种相关病毒病原体的清单，这些病原体引起与水疱相关的传染病，容易被误诊为猴痘。接下来，我们开发了一种我们称之为快速扩增子纳米孔测序（RANS）的方法。RANS 方法使用在边界和内部诊断 SNP 上具有高度同源性的诊断区域，当对这些 SNP 进行测序时，可以帮助区分组内的每个病原体。在多重 PCR 扩增过程中，同时添加 dA 尾巴和 5'-磷酸基团，从而使 PCR 产物准备好进行纳米孔测序。快速测序（几分钟）后，将读取内容与参考数据库进行比较，并识别出最近的菌株。我们首先使用在细胞系中培养的已知病毒样本测试我们的方法。所有样本都被快速准确地识别。接下来，我们检查了 2022 年猴痘疫情中的各种临床样本。我们的 RANS 方法正确识别了所有 PCR 阳性的 MPXV 样本，并将其映射到在 2022 年疫情中测序的菌株上。对于 PCR 阴性的 MPXV 样本子集，我们获得了明确的结果，鉴定出其他水疱形成病毒：人疱疹病毒 3、人疱疹病毒 2 和传染性软疣病毒。这项工作是一项概念验证研究，证明了 RANS 方法在快速和鉴别性识别一组密切相关病原体方面的潜力。我们方法的简单性和可负担性使其可以直接在任何遗传学实验室中实施。此外，其他差异诊断面板可能受益于将 RANS 方法纳入其诊断管道。
