JM-V leukemic lymphoblasts were established in cell culture. The cultured cells (JM-VLC cells) were transplantable in young chicks and produced a disease indistinguishable from JM-V lymphoblastic leukemia as initiated by whole-blood inoculation. JM-VLC cells maintained a normal female karyotype through 13 passages in Rhode Island Red cockerels. With the use of JM-V antisera and antisera from birds with naturally occurring Marek's disease (MD), specific antigens were detected on the surfaces of living cells. Intracellular antigens were detected with anti-MD virus sera after cultivation for at least 1 day at 37 degrees C. In spite of the expression of MD antigens, the presence of herpesvirus particles associated with the cultured cells, and the occurrence of foci of multinucleated cells in kidney cultures from chicks inoculated with cellfree preparations of JM-VLC cells, the pathologic potential of the cultured cells was that of JM-V leukemia.

Infection of CRFK feline kidney cells with Aleutian disease virus leads to production of virus-induced antigen(s) in the nucleus which could be demonstrated by the fluorescent-antibody technique. The number of fluorescent nuclei was linearly dependent on the dilution of the inoculum, but rarely exceeded 20% of the cells. Aleutian disease nuclear antigen was only transiently detectable. The virus-induced antigen was detected after infection of cells of several divergent species; however, the CRFK line of feline kidney cells was the most susceptible. Inhibitor studies indicated that deoxyribonucleic acid synthesis, ribonucleic acid synthesis, and protein synthesis were required for viral antigen production. Cell growth was also a requirement for synthesis of viral antigen. An in situ radioimmune assay was used to measure binding of '25I-labeled mink anti-Aleutian disease virus to infected cells and competition with unlabeled sera. The system is suitable for quantitation of infectivity. Aleutian disease (AD), a persistent viral disease of mink, has been very difficult to characterize because titration of the virus in vivo is expensive and cumbersome; often mink are naturally infected. AD antigen can be quantitated by immunodiffusion or counterimmunoelectrophoresis (CEP) (7, 8); however, the viral antigens must be laboriously purified and concentrated considerably before assaying. The virus of Aleutian disease (ADV) is a naked, icosohedral particle; estimates of the diameter of the particle have ranged from about 20 to 30 nm (6, 9, 16, 17, 21). Virus purified in CsCl gradients has been recently determined to be 27 ± 3 nm when stained with uranyl acetate and measured with internal standardization on the electron microscopic grids (17). The buoyant density of infectious virus assayed in mink and AD antigen assayed by CEP has been determined by Chesebro et al. to be 1.38 g/cm3 (6). Cho and Ingram reported a density of 1.36 g/cm3 for CEP antigen (11). Yoon et al. isolated virus at a density of 1.33 g/cm3 (21). Infectivity in mink has been shown to be resistant to lipid solvents, anionic detergents, and heat inactivation at 560C (5, 13). Infection of mink results in a large production of antibody with circulating immune complexes (18) containing low-avidity antibody (A.

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.

Properties of Aleutian disease virus (ADV) were studied using feline kidney cells, line CRFK, to assay virus by the induction of nuclear antigen. ADV nuclear antigen was detected by immunofluorescent staining. Titers of virus obtained from mink spleens at 10-8 days after infection were usually between 10(3) and 10(5) infectious units per gram of spleen. ADV was purified by fluorocarbon extraction, differential centrifugation, biogel A-15 chromatography and CsCl equilibrium centrifugation. The molecular weight of the virus was estimated to be 3-5 X 10(5) daltons. The density of antigen-inducing virus in equilibrium CsCl gradients was 1.32--1.34 g/cm3. On velocity sucrose gradients, antigen-inducing virus had a sedimentation coefficient of approximately 110S. The virus was not neutralized by sera from chronically infected mink and ferrets and by sera from experimentally infected mink. ADV was resistant to ionic and nonionic detergents and lipid solvents. The titer of partially purified virus was reduced as much as 700-fold by proteolytic enzymes but not by DNase or RNase. The virus was inactivated slowly at 56 degrees C; the initial half-life was 90 minutes. It is concluded that the properties of ADV can be determined by assay in CRFK cells, thus facilitating virological study of the disease.

Baculovirus containing the mammalianCMV promoter, in place of the insect polyhedronpromoter (BacMam), has been used to transientlytransfect COS, CHO and CHOE1a (CHO cells expressing theE1a transcriptional activator). Using this system forthe expression of a cellular adhesion factor (SAF-3) Fcfusion protein in CHOE1a, we found that levels ofexpression were highest with a MOI of 100, 20mM sodiumbutyrate, at 34 degrees C. Production increased furtherif the cells were resuspended in fresh medium, about3 x 10(6) cells ml(-1), prior to addition of the virus. These conditions were used to express 3 secretedproteins, SAF-3-Fc, CD40-hexa his and Asp 2-Fc, and, at2 to 6 days post infection, protein levels ranged from4 ug ml(-1) to 25 ug ml(-1). Based on these results, theBacMam system represents a viable technique forproducing protein at ug ml(-1) levels in a relatively shortperiod of time.