A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production.
A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production.
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DOI：
10.1186/s12896-017-0412-z
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发表时间：
2018-02-07
影响因子：
3.5
通讯作者：
Geisler C
中科院分区：
文献类型：
--
作者：
Geisler C
用于生物制品生产的细胞基质中的外源病毒污染是一个主要的安全问题。一种可用于识别外源病毒的强大新方法是生物信息学工具与大规模并行测序技术的组合。通常，这涉及针对病毒核苷酸数据库映射或BLACKS搜索单个读段。尽管这种方法对已知病毒非常敏感，但它很容易漏掉与数据库中的病毒太不相似的病毒。此外，它是计算密集型的，需要参考细胞基因组数据库。为了避免这些缺点，我们着手开发一种替代方法。我们推断，使用TBLAtron与覆盖现存病毒多样性的紧凑病毒蛋白质数据库搜索基因组和转录组组装体的外源病毒污染物，因为查询可以是快速和灵敏的，而不需要高性能计算硬件。我们在草地贪夜蛾Sf-RVN（最近分离的昆虫细胞系）上测试了我们的方法，以确定它是否被一种或多种外源病毒污染。我们使用Illumina reads来组装Sf-RVN基因组和转录组，并使用TBLAbind和我们的病毒蛋白数据库来搜索它们的外源病毒污染物。我们没有发现病毒污染的证据，这是由我们的搜索以其他方式识别编码病毒样蛋白的不同序列的事实所证实的。这些序列包括Maverick、R1 LINE和erritivirus转座子，所有这些都是昆虫基因组中常见的。我们还确定了先前描述的以及新的内源性病毒元件类似于由不同的昆虫病毒编码的ORF。我们的研究结果表明，TBLAtron搜索大规模并行测序（MPS）组件与紧凑，手动策划的病毒蛋白数据库是更敏感的外源病毒检测比BLAtron，因为我们确定了各种序列编码病毒样蛋白，但没有相似性的病毒序列在核苷酸水平。此外，搜索速度很快，不需要高性能的计算硬件。我们的研究还记录了Sf-RVN与其他Sf细胞系相比增强的生物安全性，并支持Sf-RVN非常适合生产安全生物制品的观点。本文的在线版本（doi：10.1186/s12896-017-0412-z）包含补充材料，可供授权用户使用。
Adventitious viral contamination in cell substrates used for biologicals production is a major safety concern. A powerful new approach that can be used to identify adventitious viruses is a combination of bioinformatics tools with massively parallel sequencing technology. Typically, this involves mapping or BLASTN searching individual reads against viral nucleotide databases. Although extremely sensitive for known viruses, this approach can easily miss viruses that are too dissimilar to viruses in the database. Moreover, it is computationally intensive and requires reference cell genome databases. To avoid these drawbacks, we set out to develop an alternative approach. We reasoned that searching genome and transcriptome assemblies for adventitious viral contaminants using TBLASTN with a compact viral protein database covering extant viral diversity as the query could be fast and sensitive without a requirement for high performance computing hardware. We tested our approach on Spodoptera frugiperda Sf-RVN, a recently isolated insect cell line, to determine if it was contaminated with one or more adventitious viruses. We used Illumina reads to assemble the Sf-RVN genome and transcriptome and searched them for adventitious viral contaminants using TBLASTN with our viral protein database. We found no evidence of viral contamination, which was substantiated by the fact that our searches otherwise identified diverse sequences encoding virus-like proteins. These sequences included Maverick, R1 LINE, and errantivirus transposons, all of which are common in insect genomes. We also identified previously described as well as novel endogenous viral elements similar to ORFs encoded by diverse insect viruses. Our results demonstrate TBLASTN searching massively parallel sequencing (MPS) assemblies with a compact, manually curated viral protein database is more sensitive for adventitious virus detection than BLASTN, as we identified various sequences that encoded virus-like proteins, but had no similarity to viral sequences at the nucleotide level. Moreover, searches were fast without requiring high performance computing hardware. Our study also documents the enhanced biosafety profile of Sf-RVN as compared to other Sf cell lines, and supports the notion that Sf-RVN is highly suitable for the production of safe biologicals. The online version of this article (doi: 10.1186/s12896-017-0412-z) contains supplementary material, which is available to authorized users.
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尖翅目frugiperda昆虫细胞基因组中类似色夫病毒样的内源性病毒元素被积极转录：对不定病毒检测的影响。
DOI：
10.1016/j.biologicals.2016.04.004
发表时间：
2016-07
期刊：
Biologicals : journal of the International Association of Biological Standardization
影响因子：
--
作者：
Geisler C;Jarvis DL
通讯作者：
Jarvis DL
影响因子：
5.4
作者：
Chatterjee, Atreyi Ghatak;Leem, Young Eun;Levin, Henry L.
通讯作者：
Levin, Henry L.
影响因子：
11.4
作者：
Ding, JZ;McGrath, WJ;Mangel, WF
通讯作者：
Mangel, WF
影响因子：
4.8
作者：
Hizi, A;Levin, HL
通讯作者：
Levin, HL
影响因子：
5.4
作者：
ENGELMAN, A;CRAIGIE, R
通讯作者：
CRAIGIE, R