Bradshaw G L, Schlesinger R W, Schwartz C D
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
J Virol. 1989 Apr;63(4):1704-14. doi: 10.1128/JVI.63.4.1704-1714.1989.
The responses of mouse embryo brain (MEB) cell cultures and of Madin-Darby canine kidney cells and chicken embryo fibroblasts to infection with A/PR/8/34 (PR8), A/WS/33 (WS), or the neurovirulent WSN variant were compared in terms of (i) single-cycle yields of hemagglutinating and associated neuraminidase (NA) activities and plaque-forming particles, the latter with or without trypsin activation [PFU(TR++) or PFU(TR--), respectively], and (ii) expression of nucleoprotein (NP), M1, and NS1 protein, determined for specific cell types by immunostaining, for whole culture lysates by Western blot analysis of NP and M1. Primary MEB cultures grown in serum-enriched medium were infected after 6 days (young), when none of the cells reacted specifically and exclusively with any of the nerve cell marker antibodies used, or after greater than or equal to 21 days (aged), when astrocytes (the predominant cell type), neurons, and oligodendrocytes were morphologically and immunologically mature. Secondary astrocyte-enriched cultures were used when they contained 90 to 99% of their cells as astrocytes at an early stage of differentiation. By all criteria, young MEB cultures were only marginally less permissive for each of the three viruses than were chicken embryo fibroblasts or Madin-Darby canine kidney cells. Aged MEB cultures, by comparison, produced undiminished NP, hemagglutinin, and neuraminidase, but yields of PFU(TR++) and expression of M1 protein (relative to NP) were reduced for all three viruses, most for PR8 and least for WSN; relative reduction of NS1 protein was demonstrable only in PR8-infected aged cultures. Immunostaining revealed low levels of M1 and NS1 expression only in astrocytes, not in oligodendrocytes and neurons. In PR8-infected mature astrocytes, NP accumulated in the nucleus; it persisted in some cells for at least 8 weeks after infection. The presence of NP did not seem to interfere with cell division. Secondary MEB cultures containing 90 to 99% immature astrocytes were less restricted than were aged primary cultures. Thus, it appears that reduced permissivity of nerve cell cultures, as measured in this study, is most closely correlated with advancing differentiation and maturity of astroglial cells. Assembled virions, including those that score as PFU(TR++) in restricted cultures (e.g., PR8-infected aged MEB), may be mainly products of mature oligodendrocytes and neurons.
比较了小鼠胚胎脑（MEB）细胞培养物、马-达二氏犬肾细胞和鸡胚成纤维细胞对A/PR/8/34（PR8）、A/WS/33（WS）或神经毒力WSN变异株感染的反应，具体如下：（i）血凝和相关神经氨酸酶（NA）活性以及蚀斑形成颗粒的单周期产量，后者在有或无胰蛋白酶激活的情况下[分别为PFU(TR++)或PFU(TR--)]，以及（ii）核蛋白（NP）、M1和NS1蛋白的表达，通过免疫染色针对特定细胞类型进行测定，通过对NP和M1进行蛋白质印迹分析针对全培养物裂解物进行测定。在富含血清的培养基中生长的原代MEB培养物在6天（年轻）后感染，此时没有细胞与所使用的任何神经细胞标记抗体发生特异性和专一性反应，或者在大于或等于21天（老龄）后感染，此时星形胶质细胞（主要细胞类型）、神经元和少突胶质细胞在形态学和免疫学上已成熟。当二级富含星形胶质细胞的培养物在分化早期其细胞90%至99%为星形胶质细胞时使用。根据所有标准，年轻的MEB培养物对这三种病毒中每一种的允许性仅略低于鸡胚成纤维细胞或马-达二氏犬肾细胞。相比之下，老龄MEB培养物产生的NP、血凝素和神经氨酸酶没有减少，但对于所有三种病毒，PFU(TR++)的产量和M1蛋白的表达（相对于NP）均降低，PR8降低最多，WSN降低最少；仅在感染PR8的老龄培养物中可证明NS1蛋白相对减少。免疫染色显示仅在星形胶质细胞中M1和NS1表达水平较低，在少突胶质细胞和神经元中未检测到。在感染PR8的成熟星形胶质细胞中，NP积聚在细胞核中；感染后在一些细胞中持续存在至少8周。NP的存在似乎不干扰细胞分裂。含有90%至99%未成熟星形胶质细胞的二级MEB培养物比老龄原代培养物受限制程度更低。因此，在本研究中所测定的神经细胞培养物允许性降低似乎与星形胶质细胞的分化和成熟进展最密切相关。组装的病毒粒子，包括在受限培养物中计为PFU(TR++)的那些（例如感染PR8的老龄MEB），可能主要是成熟少突胶质细胞和神经元的产物。
