McClenahan Shasta D, Uhlenhaut Christine, Krause Philip R
Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.
Highly Pathogenic Viruses, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Berlin, Germany.
Vaccine. 2014 Dec 12;32(52):7115-21. doi: 10.1016/j.vaccine.2014.10.022. Epub 2014 Oct 27.
We employed a massively parallel sequencing (MPS)-based approach to test reagents and model cell substrates including Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), African green monkey kidney (Vero), and High Five insect cell lines for adventitious agents. RNA and DNA were extracted either directly from the samples or from viral capsid-enriched preparations, and then subjected to MPS-based non-specific virus detection with degenerate oligonucleotide primer (DOP) PCR. MPS by 454, Illumina MiSeq, and Illumina HiSeq was compared on independent samples. Virus detection using these methods was reproducibly achieved. Unclassified sequences from CHO cells represented cellular sequences not yet submitted to the databases typically used for sequence identification. The sensitivity of MPS-based virus detection was consistent with theoretically expected limits based on dilution of virus in cellular nucleic acids. Capsid preparation increased the number of viral sequences detected. Potential viral sequences were detected in several samples; in each case, these sequences were either artifactual or (based on additional studies) shown not to be associated with replication-competent viruses. Virus-like sequences were more likely to be identified in BLAST searches using virus-specific databases that did not contain cellular sequences. Detected viral sequences included previously described retrovirus and retrovirus-like sequences in CHO, Vero, MDCK and High Five cells, and nodavirus and endogenous bracovirus sequences in High Five insect cells. Bovine viral diarrhea virus, bovine hokovirus, and porcine circovirus sequences were detected in some reagents. A recently described parvo-like virus present in some nucleic acid extraction resins was also identified in cells and extraction controls from some samples. The present study helps to illustrate the potential for MPS-based strategies in evaluating the presence of viral nucleic acids in various sample types, including cell culture substrates and vaccines.
我们采用了基于大规模平行测序（MPS）的方法，来测试包括中国仓鼠卵巢（CHO）、Madin-Darby犬肾（MDCK）、非洲绿猴肾（Vero）和High Five昆虫细胞系在内的试剂和模型细胞底物是否存在外源因子。RNA和DNA要么直接从样品中提取，要么从富含病毒衣壳的制剂中提取，然后使用简并寡核苷酸引物（DOP）PCR进行基于MPS的非特异性病毒检测。对独立样本比较了454、Illumina MiSeq和Illumina HiSeq的MPS结果。使用这些方法可重复实现病毒检测。来自CHO细胞的未分类序列代表尚未提交到用于序列鉴定的常用数据库的细胞序列。基于MPS的病毒检测灵敏度与基于细胞核酸中病毒稀释的理论预期限度一致。衣壳制剂增加了检测到的病毒序列数量。在几个样本中检测到了潜在的病毒序列；在每种情况下，这些序列要么是人为的，要么（基于进一步研究）显示与具有复制能力的病毒无关。在使用不含细胞序列的病毒特异性数据库进行的BLAST搜索中，更有可能鉴定出病毒样序列。检测到的病毒序列包括先前在CHO、Vero、MDCK和High Five细胞中描述的逆转录病毒和逆转录病毒样序列，以及在High Five昆虫细胞中的诺达病毒和内源性杆状病毒序列。在一些试剂中检测到了牛病毒性腹泻病毒、牛hokovirus和猪圆环病毒序列。在一些样本的细胞和提取对照中还鉴定出一种最近在一些核酸提取树脂中描述的细小病毒样病毒。本研究有助于说明基于MPS的策略在评估各种样本类型（包括细胞培养底物和疫苗）中病毒核酸存在情况方面的潜力。
