Human endothelial cells derived from the umbilical vein were transformed with SV40 virions. A cell line subcultured for over 60 serial passages was characterized in comparison with its untransformed counterpart which was culturable for less than five passages.The SV40-transformed human endothelial cells, designated SV-HUVEC, were positive not only for tumor (T) antigen specific to the SV40-transformed cell, but also for two markers of endothelial cells, Factor VIII-related antigen and a receptor for Ulex europaeus agglutinin I. By transformation the growth potential of the human endothelial cells was increased and their serum requirement was decreased. The SV40-transformed endothelial cells were, however, unable to form colonies in soft agar or to form tumors in athymic nude mice, although a small nodule was produced at the site of inoculation. Subcultivation of these cells up to the 62nd passage eventually resulted in crisis and loss of further cell division. Thus, the human endothelial cells were transformed by SV40 while retaining certain normal functions but without showing tumorigenicity.Endothelial cells cover all the vascular walls in a one-wall-thick layer and form a continuous lining between the circulating blood and the surrounding tissues. Endothelial cells also play an important role in wound healing (19), the reendothelialization of vessel-wall injury (23), and tumor growth (1, 3). Establishment of a continuous cell line of endothelial cells would facilitate studies on the physiologic and pathologic factors that induce endothelial mitosis. In addition, transformation of the endothelial cells may offer a clue to a better understanding of tumors of endothelial origin, such as Kaposi's sarcoma (4, 11).Transformation of human endothelial cells with SV40 was first accomplished by Gimbrone and Fareed, in 1976, by transfection with intact circular DNA or linear fragments containing the entire early-gene region, but not by infection with SV40 virions. However, the transformed endothelial cells were negative for Factor VIIIrelated antigen (FVIII-RAG) (6).Recently, transformation of endothelial cells by infection with SV40 virions was achieved and a cell line positive for FVIII-RAG was subcultured up to the 62nd passage in our laboratory. The characteristics of the SV40-transformed endothelial cells, designated SV-HUVEC, are described in this paper.

A case of progressive multifocal leukoencephalopathy (PML) reported previously to be of simian virus (SV40) etiology was re‐evaluated. The supernatant from a 10% homogenate of brain material was inoculated into African green monkey kidney cells and BSC‐1 cells which are permissive for SV40. However no cytopathic effect (CPE) developed and no virus was isolated. The brain supernatant agglutinated human group O erythrocytes and contained 5120 units/mL. The Hirt supernatant from the brain contained three DNA bands corresponding to forms I, II and III of circular double‐stranded viral DNA. Restriction endonuclease cleavage analysis revealed that this viral DNA was different from SV40 DNA, but similar to JC virus DNA. After cloning of this viral DNA into pBR322 at the BamHI site, DNA homology of this virus and of SV40 was investigated. The cloned DNA hybridized with all four HpaI/EcoRI fragments of SV40 genome at the effective temperature (Tm) of −50°C. At Tm −28°C, however, the cloned DNA hybridized with only HpaI/EcoRI fragment B of the SV40 genome. In contrast to this, JC virus DNA hybridized with all five EcoRI/BamHI/HindIII fragments of cloned DNA even at Tm −28°C. Therefore, the causative agent of this PML case was not SV40 but JC virus.

The interaction between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and two neural cell lines, mouse neuroblastoma (N1E-115) and rat glioma (C6-BU-1), was investigated. N1E-115 cells were permissive to both types of HSV. In C6-BU-1 cells, on the other hand, all the HSV-1 strains tested so far showed persistent infection, and the infectious virus of HSV-2 strains disappeared spontaneously. The HSV-2-infected C6-BU-1 cells were positive for HSV-2-specific DNA sequences, virus-specific RNA, HSV-2-specific antigens and thymidine kinase activity, when no infectious virus was detected. The HSV-2 was reactivated from those C6-BU-1 cells by superinfection with murine cytomegalovirus (MCMV), but not with UV-irradiated MCMV or human cytomegalovirus. The reactivated. HSV-2 was identical to the parental virus, when examined by restriction endonuclease cleavage analysis.After primary infection, herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) cause latent infections in neural tissues and no infectious virus can be isolated under such condition. The latent HSV, however, can be reactivated by various stimuli to neuron (39) or to the skin in vivo (5, 14, 31) or can be retrieved by explant culture of the ganglion in vitro (3,33).To analyze this unique virus-cell interaction, various models have been established in vitro. The in vitro latency models most analyzed to date depend on the presence of various virus inhibitors for establishment and incubation at supraoptimal temperature for maintenance of the latency (6,7,(40)(41)(42)(43)(44). Exceptionally, in a lymphoblastoid T cell line (13) and a hyperresistant neuroblastoma cell line (23), HSV-1 became latent without any artificial manipulation.In contrast to HSV-1, only a few latency models for HSV-2 have been developed in vitro. These included latency of HSV-2 in human fetal fibroblasts (24), human embryonic lung cells (6,7,29), and human neuroblastoma cell line (47). All the in vitro models for HSV-2 were developed under artificial conditions similar to HSV-1, and no spontaneous latency model was established.In this paper, we describe the interaction between HSV-2 and a rat glioma cell line (C6-BU-1), in which infectious virus disappeared spontaneously, but reactivated by superinfection with murine cytomegalovirus (MCMV).

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