DETECTION OF VIRUSES IN CELL SUBSTRATES

CBER感兴趣的是细胞系是否用于产品生产 包含病毒或类似病毒的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。在以前的 研究中，我们使用电子显微镜检查内源性逆转录病毒- 类似于在中国仓鼠卵巢(CHO)细胞系中发现的颗粒 被用作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 在电子显微镜下可见。我们试图确定是否 生长因子或细胞因子(可能在CHO细胞中产生) 可能会影响这些内源颗粒。结果表明， CHO细胞处理后颗粒物从30%-100%增加 几种细胞因子(GM-CSF、干扰素、肿瘤坏死因子)或与佛波酯TPA。一个 描述这些发现的手稿已发表在《体外》杂志上 毒理学(1991)。我们最近接触了另一种细胞底物 问题。由于许多新的重组生物制品被生产出来 利用杆状病毒表达系统和昆虫细胞系 被称为Sf9；而对其他可以 感染Sf9细胞，特别是那些具有人类宿主范围的细胞，我们最近 启动了一项研究，以确定是否有任何对人类有致病作用的病毒 可以感染Sf9细胞。我们已经检查了一系列病毒的 感染Sf9细胞的能力，以及评估Sf9是否 细胞没有病毒污染，适合用于 生物制品生产得到了发展。我们评估了 15种精选病毒感染Sf9细胞的能力 代表几类虫媒病毒以及其他几种 宿主范围广的病毒。圣路易斯脑炎病毒(SLE) 黄病毒可以高效地感染SF9细胞。结果有三个 其他黄病毒，黄热病减毒株(17D疫苗 株)(YF)，登革病毒1(DEN-1和2(DEN-2)，提示可能较低 不能持续的水平感染。系统性红斑狼疮导致Sf9的CPE 细胞(10-20%巨细胞)。YF、DEN-1和DEN-2不产生明显的 Sf9细胞中的CPE。在SF9细胞中也可以通过以下方法检测到SLE 免疫荧光和电子显微镜。Vero细胞上的空斑分析 使用SLE感染的Sf9细胞的过滤上清液，结果为阳性。 此外，我们测试的许多病毒显然可以存活 在长时间的细胞培养中不会引起明显的CPE，几个可以 仅在媒体中就能存活长达8周。这可能带来以下风险 Sf9电池生产的产品的污染(如果适当的测试) 是不成立的。

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.