DETECTION OF VIRUSES IN CELL SUBSTRATES

细胞基质中病毒的检测

• 批准号: 3792523
• 负责人: C J MARCUS-SEKURA
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3792523
• 关键词: Baculoviridae; CHO cells; Flaviviridae; biological products; clone cells; colony stimulating factor; dengue virus; electron microscopy; encephalitis virus; genetic manipulation; immunofluorescence technique; interferons; nucleic acid sequence; phorbols; plaque assay; tumor necrosis factor alpha; virus classification; virus cytopathogenic effect; virus infection mechanism; virus replication; viruslike particle

It is of interest to CBER whether cell lines used for product production contain virus or virus-like particles or viral nucleic acid sequences or support the growth of viruses infectious for humans. In previous studies, we used electron microscopy to examine endogenous retroviral- like particles found in the Chinese Hamster Ovary (CHO) cell line which is used as the cell substrate for several recombinant products made in CHO cells. CHO cells contain endogenous retroviral-like particles visible by electron microscopy. We attempted to determine whether growth factors or cytokines (which might be manufactured in CHO cells) might affect these endogenous particles. The results indicated increases in particles from 30-100% after treatment of CHO cells with several cytokines (GM-CSF, IFN, TNF) or with the phorbol ester TPA. A manuscript describing these findings has been published in In Vitro Toxicology (1991). We have recently approached another cell substrate issue. Since many new recombinant biological products are produced using the Baculovirus expression system and use an insect cell line called Sf9; and relatively little is known about other viruses which can infect Sf9 cells, particularly those with human host range, we recently initiated a study to determine whether any viruses pathogenic for humans can infect Sf9 cells. We have examined a series of viruses for their ability to infect Sf9 cells, and protocols for evaluating whether Sf9 cells are free of viral contamination and suitable for use for production of biological products have been developed. We evaluated the ability of Sf9 cells to be infected by fifteen viruses chosen to represent several classes of arboviruses as well as several additional viruses with wide host range. St Louis Encephalitis virus (SLE) a flavivirus, can productively infect the SF9 cells. Results with three other flavivirus, attenuated strains of Yellow Fever (17D vaccine strain) (YF), Dengue virus 1 (DEN-1 and 2 (DEN-2), suggest possible low level infection which cannot be sustained. SLE causes CPE in the Sf9 cells (10-20% giant cells). YF, DEN-1, and DEN-2 do not produce obvious CPE in the Sf9 cells. SLE can also be detected in SF9 cells by immunofluorescence and electron microscopy. Plaque assays on Vero cells using filtered supernatants from SLE-infected Sf9 cells are positive. Furthermore, many of the viruses we tested apparently can survive for long periods in cell culture without causing apparent CPE, several can survive up to 8 weeks in media alone. This may present risks of contamination for products produced in Sf9 cells if appropriate testing is not instituted.

CBER感兴趣的是用于产品生产的细胞系是否 含有病毒或病毒样颗粒或病毒核酸序列，或 支持对人类具有传染性的病毒的生长。 前几 研究中，我们用电子显微镜检查内源性逆转录病毒， 在中国卵巢癌（CHO）细胞系中发现的类似颗粒， 用作生产的几种重组产品的细胞底物， CHO细胞。 CHO细胞含有内源性逆转录病毒样颗粒 通过电子显微镜可见。 我们试图确定 生长因子或细胞因子（可能在CHO细胞中生产） 可能会影响这些内源性颗粒。 结果表明 在用以下物质处理CHO细胞后，颗粒从30-100%增加 几种细胞因子（GM-CSF、IFN、TNF）或与佛波醇酯TPA。一 描述这些发现的手稿已发表在《体外》杂志上 毒理学（1991年）。 我们最近研究了另一种细胞基质 问题. 由于许多新的重组生物制品被生产出来 使用杆状病毒表达系统和使用昆虫细胞系 称为Sf 9;对其他病毒的了解相对较少， 感染Sf 9细胞，特别是那些与人类宿主范围，我们最近， 发起了一项研究，以确定是否有任何病毒致病的人类 可以感染Sf 9细胞。我们已经研究了一系列病毒， 感染Sf 9细胞的能力，以及评估Sf 9是否 细胞没有病毒污染，适合用于 生物制品的生产得到了发展。 我们评估了 Sf 9细胞被15种病毒感染的能力， 代表了几类虫媒病毒以及几种其他的 宿主范围广的病毒。圣刘易斯脑炎病毒（SLE）a 黄病毒，可以生产性地感染SF 9细胞。 三个结果 其他黄病毒黄热减毒株（17 D疫苗 菌株）（YF）、登革病毒1型（DEN-1和2型（DEN-2），提示可能低 无法持续的水平感染。 SLE导致Sf 9中的CPE 细胞（10-20%巨细胞）。 YF、DEN-1和DEN-2不产生明显的 Sf 9细胞中的CPE。 SLE也可以在SF 9细胞中检测， 免疫荧光和电子显微镜。Vero细胞上的噬斑试验 使用来自SLE感染的Sf 9细胞的过滤的上清液进行的检测是阳性的。 此外，我们测试的许多病毒显然可以存活 在细胞培养中长时间不引起明显的CPE，几种可以 在单独的培养基中存活长达8周。 这可能会带来以下风险： 在Sf 9细胞中生产的产品的污染（如适用）检测 并不是建立起来的。