Passage In Tissue Culture Research Articles

Isolation and characterization of an endogenous type C RNA virus of mink (Mv1Lu) cells.

Cells of the established MvlLu mink line spontaneously released a reverse transcriptase-containing virus after long-term passage in tissue culture. By molecular hybridization, DNA of normal mink cells was found to possess extensive nucleotide sequence homology with a reverse-transcription product of the viral genome, demonstrating that the new isolate was an endogenous virus of mink origin. The mink virus shared antigenic determinants with the major structural proteins of known mammalian type C viruses. Double-antibody competition radioimmunoassays were developed by utilizing the purified major structural protein, p30, of the mink endogenous virus. The virus was shown to possess antigenic determinants unique from those of other known mammalian type C viruses. It exhibited a higher degree of immunological cross-reactivity with endogenous rat type C and horizontally transmitted feline leukemia viruses than with other mammalian type C viruses tested. The finding that mink cells can remain nonvirus producing for many cell generations argues that there normally exists some cellular restriction to endogenous virus expression in this species.

Morphologic and histochemical characteristics of cell lines derived from hamster peripheral lung tumors

Intratracheal instillation of the alpha emitter polonium- 210 ( 210 Po) or benzo[a]pyrene induces peripheral lung tumors in Syrian hamsters which have a combined epidermoid and adenomatous morphology similar in appearance to human bronchiolar-alveolar carcinoma. Two permanent cell lines derived from such tumors have been studied in an attempt to determine the cell of origin of the original peripheral tumors. The ultrastructural and histochemical characteristics of both the cells and of cheek pouch tumors grown from HLAC- 4 and HLAC- 14 cells at various stages of passage in tissue culture in general support, but do not prove, our hypothesis that Clara cells found in the terminal and respiratory bronchioles are the cells of origin of the peripheral lung tumors induced in hamsters by these carcinogens.

Functional-morphological Correlates of Renomedullary Interstitial Cells

1. Indomethacin treatment of renomedullary interstitial cells (RIC) causes a marked increase in number and size of their lipid droplets. The anti-hypertensive function is retained. 2. After multiple passages in tissue culture, RIC lose their ability to produce an anti-hypertensive effect in hypertensive animals. Along with this functional loss, lipid droplets and a prominent cisternal system become attenuated or disappear. 3. We conclude that preservation of the lipid granule-cisternal organelle relationship is important in preservation of the anti-hypertensive endocrine function of RIC.

VIROLOGIC CONSIDERATIONS ON ANTI-DENGUE VACCINE

Possibilities of developing an anti-dengue vaccine are discussed based on our experimental findings with a dengue virus type 1 (DEN-1) Mochizuki strain. The Mochizuki strain virus was attenuated for human beings in the course of several passages through mouse brains since its isolation from the blood of a dengue patient in the 1943 epidemic in Nagasaki, Japan. This attenuated virus in the living state induced no abnormal symptoms after injection into human volunteers or monkeys, yet it stimulated production of specific anti-DEN-1 NT and HI antibodies and induced a prophylactic effect on the human beings against virulent dengue viruses (in the form of patient's serum) given experimentally. The immunogenic effects of the virus was maintained after passages in primary monkey kidney tissue cultures. Using the tissue culture virus, together with the 17D yellow fever (YF) vaccine, a combined immunization of human beings against DEN and YF was possible; production of the antibodies of significant titers against both viruses was unequivocally demonstrated. The partially purified virus was similarly effective in stimulating production of the anti-DEN antibodies, and the same statements could be made as to the viral components obtained by sucrose density gradient centrifugation. The virus was shown to multiply in the WI-38 cell line cultures, and this suggests application of this cell line to produce a live tissue culture vaccine.Conclusions of this review are : (1) the Mochizuki virus is completely attenuated; (2) yet it stimulates production of specific anti-DEN antibodies and induces protection against DEN-1 infection by virulent human virus; and (3) that possibility of developing a live tissue culture vaccine is suggested; this also makes possible a combined DEN-YF vaccination on the experimental basis. It is stated in the connection that the purified virus or its components can be used for the above purposes.Problems regarding the implication of “hypersensitivity hypothesis” of dengue and dengue hemorrhagic fever-shock syndrome in anti-DEN vaccination are in addition discussed.

Chapter 30 Selective Cultivation of Mammalian Epithelial Cells

Epithelial cells are often recognizable in primary cell cultures, but are usually overgrown in later subcultures by the more rapidly dividing fibroblastic cells from connective tissue. Several studies in which cultures were attempted from 100 or more epithelial tumors reported success rates of only 5% or less. Normal human epithelial cells, capable of serial subculture through varying numbers of generations, have been cultured from brain, mesothelium, and endothelium. In recent years, a few reports have described methods for selective culture of epithelial cells from normal or neoplastic tissues of rats, mice, or hamsters. Some of these methods do not completely eliminate fibroblasts, while others employ complex technical procedures (e.g., organ perfusion, cloning, alternate tissue culture and animal passages, mechanical destruction of undesired cell types), which limit their general usefulness.

Transmissible mink encephalopathy: infectivity of corneal epithelium.

Corneal epithelium from hamsters dying of transmissible mink encephalopathy contained a virus titer of 10-4.8 times the 50 percent lethal dose (10-4.8 LD50) per 0.05 milliliter when assayed as a cell suspension derived directly from the infected animal. After one passage in tissue culture, an equivalent concentration of cells contained only 10-0.8 LD50 per 0.05 milliliter.. It is concluded that corneal tissues are infectious; the infectivity may be mainly associated with free nerve endings. However, the most important immediate inference is that corneas from human beings affected with Creuzfeldt-Jakob disease are likely to be lethal if transplanted to healthy recipients.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. Transformation of hamster kidney cells by adenovirus 2 and the nondefective hybrid viruses.

Nonhybrid adenovirus 2 (Ad2) and five nondefective Ad2-simian virus 40 (SV40) hybrids (Ad2(+)ND(1)-Ad2(+)ND(5)) failed to produce tumors when injected into newborn hamsters. However, each of these agents transformed hamster kidney cells in vitro, and the transformed cell lines produced tumors when injected into suckling hamsters. Foci of cells transformed by the hybrids could not be distinguished from foci of cells transformed by nonhybrid Ad2. Histopathologically, tumors induced by the nondefective hybrid-transformed lines were of the adenovirus type with no areas of SV40-type fibrosarcoma. All of the cell lines studied contained Ad2 T antigen and Ad2 RNA. Only the B55HK(1) line contained the three early SV40 antigens, U, TSTA, and T. The ND(4)HK(1) line contained SV40 T and TSTA antigens (the latter antigen being detected only after 48 to 49 tissue culture passages) and SV40 RNA. The ND(2)HK(2) line contained SV40 RNA but no detectable SV40 antigens; the other hybrid-transformed lines contained no SV40 RNA. The transformation of hamster cells by the nondefective Ad2-SV40 hybrids seemed to be caused by the Ad2 genome and could not be attributed to the regions of the SV40 genome contained within the hybrids. Additionally, these results indicate that interactions between the genomes of the nondefective hybrid viruses and the hamster cells they transform are complex and could involve the site of integration, the size of the incorporated viral genome, the transcription or translation of the viral genome, or various combinations of these processes.

Electron microscopic identification of papova virus in laryngeal papilloma.

AbstractAttempts to propagate a laryngeal papilloma virus in cell culture by the establishment of tissue culture lines from 27 papilloma specimens which were removed endoscopically from the larynx of 19 patients of all age groups were unsuccessful. By light microscopy, all the lesions, with one exception, exhibited the usual features of a typical squamous papilloma. Laryngeal papillomas were removed endoscopically from an adult male Caucasian on three separate occasions with intervening intervals of 13 and seven months. Tissue removed at each procedure was submitted for study. Tumor obtained from the first two operative procedures exhibited the features of a benign squamous papilloma. Histologic diagnosis of the most recent excised laryngeal neoplasm was consistent with carcinoma.Two papillomas which contained papova‐like virus when examined by electron microscopy were among the excised specimens cultured. Laryngeal papillomas were removed endoscopically on two occasions at an interval of five months from a 22‐year‐old Caucasian male who had a one‐month history of hoarseness. Electron microscopic examination of the most recently excised tissue revealed intranuclear viral particles which were in the shape of an icosahedron and measured approximately 45 mμ in diameter. The particles contained a densely staining core and were generally scattered throughout the nucleus. Occasionally, they were seen in crystalline arrays. The nuclear site of replication, their size and shape fit the description of the agents belonging to the papova virus group. Papova‐like viruses were also seen in laryngeal papillomas from an 18‐month‐old Negro child born of a mother with venereal warts.Although many attempts at propagating the human papilloma virus have been reported,1 the virus known to cause common warts has to date never been successfully grown for continuous passage in tissue culture. The difficulty in isolating the laryngeal papilloma virus in tissue culture is also apparent.2Serial studies done on an adult laryngeal papilloma showed transition of the tumor from a benign to a malignant lesion. It is interesting to speculate on this possibly viral‐induced laryngeal malignancy. That viruses can cause malignancy in non‐human species is well known, and there is no reason to presume that man is different from such species in this respect. Clinical evidence of the transmissability of this disease from infected genital warts to the newborn was suggested by the development of laryngeal warts in the young child. The electron microscopic identification of virus in the laryngeal papilloma that is morphologically similar to the genital wart virus, supports this theory of transmissability. The ultrastructure of human laryngeal papilloma has been described previously3 and the presence of virus4 and virus‐like particles suggested;5 however, the crystalline lattice assemblies, characteristic of the papova group, have never been described heretofore. The results of these studies add strong morphologic and clinical evidence of the viral etiology of laryngeal papilloma; however, the evidence is not conclusive. The ultimate aim of this project is to propagate the papilloma virus in tissue culture.

Molecular probes in studies of the relationship of viruses to human neoplasia.

Extensive studies in a number of laboratories have established the role of ribonucleic acid (RNA) viruses in the origin of animal neoplasms of diverse types. Recent studies have characterized the viruses biochemically and have developed biochemical tools for study of human tumors. These technics have been applied to an analysis of the role of viruses in human solid tumors and leukemia. In a series of studies based on the animal leukemia–sarcoma virus model system, cultures derived from human fibrosarcoma desmoides and from giant cell tumor of bone were inoculated with fresh bone marrow aspirates from patients with acute, lymphocytic leukemia. Foci of morphologically altered cells appeared in these cultures several tissue culture passages after inoculation. Cell lines were derived from the altered cells and were characterized biologically and biochemically. The cells exhibited growth properties and tumorigenicity characteristic of malignant cells in immunosuppressed mice. The cells were also found to release particles (buoyant density 1.15 to 1.17 Gm. per ml.) containing reverse transcriptase and high molecular weight RNA. The data obtained suggest that genetic information similar to that of animal RNA tumor viruses was present in the cells and was activated during transformation. The results add further evidence implicating RNA tumor viruses in the etiology of some types of human neoplasia, such as leukemia, giant cell tumor, and fibrosarcoma desmoides.

A stable freeze-dried smallpox vaccine made in monolayer cultures of primary rabbit kidney cells

Smallpox vaccine was made in monolayers of primary rabbit kidney cells. The seed virus used for the production of this vaccine was the same as used for the production of calf lymph so that the vaccine contained vaccinia virus with not more than one tissue culture passage. As to other aspects this vaccine also met the international requirements for safety, potency and stability. For the lyophilization of this tissue culture smallpox vaccine a number of stabilizers routinely used for the lyophilization of several products were tested. A highly stable vaccine was obtained with 5% peptone-5% sorbitol for both the tissue culture smallpox vaccine and the calf lymph and the addition of other stabilizers did not increase the stability. With the same combination of stabilizers and lyophilized tissue culture smallpox vaccine, whether the ampoules were sealed in vacuo or in nitrogen, turned out to be more stable than lyophilized calf lymph. Cell cultures from approximately 500 rabbits were tested for adventitious agents. No cytopathic changes were observed and no viruses or other micro-organisms were isolated.

Differing neurovirulence of three Sabin attenuated type 3 vaccine seed pools and their progeny

A comparison was made of three pools of virus prepared from the type 3 Sabin original poliovirus. A similar study of commercial and experimental harvests made from them revealed differences related to tissue culture passage and seed. The significance of these findings for the production of commercial oral attenuated poliovirus vaccines is discussed.

Properties in cell cultures of a hamster brain-adapted subacute sclerosing panencephalitis virus.

Several viruses antigenically indistinguishable from measles virus have been isolated from the brains of patients with subacute sclerosing panencephalitis (SSPE), a progressive neuropathy of children, by co-cultivation of brain cells with other cell types (Horta-Barbosa et al. 1969a; Payne, Baublis & Itabashi, 1969). The SSPE-1 virus isolated by Horta-Barbosa et al. (1969b) was obtained as second passage fluids from Dr Wolfgang Zeman, Indiana University Medical Center. In our laboratory, SSPE-1 virus (mantooth strain) was adapted to the brains of newborn hamsters by four pairs of alternate passages in hamster brain and monkey cell tissue culture (Byington, Castro & Burnstein, 1970). This communication presents some of the characteristics of the hamster-brain-adapted (HBS) virus in cell culture. Stock virus was the 10th passage from the original brain isolate. It was prepared by intracerebral inoculation of neonatal hamsters with 0.02 ml. of HBS virus; affected brains were harvested at 96 hr, when pronounced neurological signs were evident.

A large and small plaque variant of sindbis virus obtained by serial passages in tissue culture of chick embryo cells.

SummaryA large plaque variant of Sindbis virus was obtained by repeatedly cloning after serial passage 50 times in chorioallantoic membrane in tissue culture. This virus has a high infectious titer in CEF cells as well as suckling mice. On the other hand, a small plaque of Sindbis virus obtained by the same method above using chick embryo brains instead of chorioallantoic membrane, grows to somewhat lower titer than that of the large plaque variant, and is not sensitive to agar inhibitors.

Neutralizing Activity of Sera from Guinea-Pigs Inoculated with Foot-and-Mouth Disease Virus Variants

Summary The neutralizing activity of sera from guinea-pigs inoculated with small-plaque (SP) and large-plaque (LP) variants of foot-and-mouth disease virus (FMDV) was studied. Virus neutralization tests were performed in suckling mice using the SP and LP variants as well as a low passage virus of the same type and strain. Higher titres were obtained with homologous virus than when a vims of low passage in tissue culture was used. Neutralizing activity was not demonstrable in some guinea-pig sera with a low passage virus, whereas a significant level of neutralizing activity was detected when homologous virus-serum mixtures were tested.

Continuous Type C Virus Production in a Tissue Culture of Human Origin (Burkitt Lymphoma)

Electron microscopy of numerous tissue cultures derived from various types of human tumors revealed only occasional type C virus particles in tissue cultures of leukemia and lymphoma (Dmochowski, L., Fourteenth Annual Conf. on Cancer, Yearbook Medical Publ, Inc., 37, 1969) and of solid tumors such as osteosarcoma, giant cell tumors (Priori, E. S. et al., J. Nat. Cancer Inst., 1971, in press), and in rhabdomyosarcoma.This report describes a monolayer tissue culture derived from pleural effusion cells of a patient with Burkitt lymphoma (American type) which continuously produces type C virus particles. The child expired 14 days after collection of the pleural effusion due to a generalized varicella infection. Varicella virus particles could not be found in the original pleural effusion cells nor in any of the tissue culture passages.

Neoplasms of the Hematopoietic System in Nonhuman Primates: Report of One Spontaneous Tumor and Two Leukemias Induced by Procarbazine<xref ref-type="fn" rid="FN2">2</xref>

Three neoplasms of the hematopoietic system in nonhuman primates are reported. A spontaneous reticulum cell sarcoma arose in an adult African green monkey (Cercopithecus aethiops), and myelogenous leukemias were induced in 2 female rhesus monkeys (Macaca mulatta) at ages 17 months and 5½ years after treatment with procarbazine hydrochloride. Transplantation of the reticulum cell sarcoma to progeny of the donor and the myelogenous leukemias to newborn rhesus monkeys was unsuccessful. No virus particles were detected by electron microscopy, and no viruses were isolated after tissue culture passage.

Properties of transformed hamster cells containing sv40 tumor antigen in the cytoplasm

AbstractThe properties of hamster cells containing SV40 tumor (T) antigen in the cytoplasm, rather than the nucleus, were determined. Eight cell lines were established from eight tumors induced by hamster embryo fibroblasts transformed in vitro by PARA (2cT)‐adenovirus 7. Six cell lines contained only cytoplasmic SV40 T‐positive cells while two were a mixture of nuclear T‐positive and cytoplasmic T‐positive cells. All the cell lines contained SV40 S antigen, all caused the production of SV40 T antibody in vivo, and four elicited the production of adenovirus T antibody. The cell lines seem to have acquired an infinite life span in vitro. The localization of T antigen apparently can be a stable phenomenon because five of the cell lines have retained T antigen exclusively in the cytoplasm for over 40 passages in tissue culture. The cytoplasmic T antigen could be detected by complement fixation in addition to immunofluorescence. Cytoplasmic T‐positive cells were readily transplantable in vivo and contained SV40 TSTA demonstrable by both immunogenicity and immunosensitivity procedures. Two of the three cytoplasmic variants of PARA appear to be weakly oncogenic in newborn hamsters.

3. Antigenic relationships of mosquito cell lines as determined by immunodiffusion techniques.

Studies were undertaken in our laboratories to identify mosquito cell lines and/or to determine the antigenic relationships among them by gel precipitin techniques using both immunodiffusion and immunoelectrophoresis. Table 6 shows the list of monolayer and suspended cell cultures used in this study, the primary source from which they were derived, and the tissue culture passage level.KeywordsInsect Cell LineAntigen PreparationInsect Cell CultureAntigenic ComponentWalter Reed Army InstituteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Experimental infection of Aedes aegypti and Aedes albopictus with dengue viruses.

The susceptibility of two Aedes (Stegomyia) species, Ae. aegypti and Ae. albopictus, to infection with dengue virus was assessed using both natural feeding on a viraemic gibbon and a membrane feeding technique. Both species were highly susceptible to dengue virus infection. It was found that both species were more sensitive than the LLC-MK2 cell plaque assay system used in detecting viraemia in a gibbon infected with a low tissue culture passage strain of dengue-2 virus. However, the same strain passaged in LLC-MK2 cells failed to infect either species when the mosquitoes were fed upon a suspension of this virus. Both species were infected readily when fed upon a suspension of a mouse adapted strain of dengue-2 virus.

Experimentally Induced Alterations in Antigenic Properties of an Arbovirus Serotype

Antigenic properties of Getah virus were monitored during 80 serial passages in Vero tissue cultures. The parent virus lost its serotype identity by passage 80. A clone derived from the parent remained antigenically stable during passage. The experimentally induced antigenic alterations in Getah virus were attributable to the tissue culture host selection of constituent subpopulations of virus with properties unlike those of the original parent. The marked differences in neutralization titers of low- and high-passage virus were considered related to or influenced by (i) the variation in subpopulation selection of the virus during passage and (ii) the variation in availability of antigenic stimulants during preparation of antiserum.