Studies on the propagation in vitro of poliomyelitis viruses. I. Viral multiplications in tissue cultures employing monkey and human testicular cells.
Poliomyelitis virus was propagated in vitro successfully in extraneural tissues. Suspended tissue fragment cultures and combined plasma clot-suspended tissue fragment cultures of monkey or human testicular tissues were employed. Five strains representative of poliomyelitis virus were maintained for from 36 to 263 days in the suspended tissue fragment type of culture. The dilution factors calculated by tissue replacements for the eight serial passages ranged from 10(7.8) to 10(44.5) and when assessed by fluid replacements, from 10(15) to 10(95.3). The LD(50) for each strain of Type 2 virus was determined for selected transfers. The identify of each strain of virus was established by neutralization tests and histopathological findings in monkeys dead from the injection of tissue culture virus. Control experiments and other tests made known that propagation of poliomyelitis virus did not occur in the absence of viable testicular cells and that an extraneous virus was not inadvertently acquired during the course of these studies. (+info)
Studies on the propagation in vitro of poliomyelitis viruses. II. A description of the growth curve and its relation to the cytopathogenicity of poliomyelitis virus.
The growth of poliomyelitis virus, Type 2, Yale-SK strain, in cultures of monkey testicular tissue was observed to occur in discrete cycles. Growth curves showed that each cycle was composed of (a) an initial lag phase when little or no virus was released from the cells, (6) a phase of viral production, and (c) a plateau which represented a decrement in the rate of viral production. This pattern of viral multiplication occurred in monkey testicular tissue cultures which have as the liquid phase either ox serum ultrafiltrate or monkey serum-chicken embryonic extract medium. The presence of a solid medium composed of chicken plasma, clotted either with chicken embryonic extract or bovine thrombin, did not alter the pattern of viral multiplication. The shape of the growth curve as established by any of four different techniques for tissue cultivation, was shown to be independent of the cultural technique employed. For cultures of monkey testicular tissue, the amount of virus in the tissue was as much as tenfold greater than that in the liquid of the same cultures. Moreover, viral production was evident earlier and was detectable for a longer period of time in the tissue than in the liquid phase. The rapidly incremental phase of the growth cycle, when large quantities of virus were released into the liquid phase, coincided in time with the destruction of the spindle-shaped cells, which extended from the explants. Although destruction of outgrowth cells was marked, there remained cells within the explants capable of supporting the growth of poliomyelitis virus. (+info)
Studies on the propagation in vitro of poliomyelitis viruses. III. The propagation of poliomyelitis viruses in tissue cultures devoid of nerve cells.
Cells like fibroblasts, having no resemblance whatever to nerve cells were obtained in morphologically pure cultures from monkey testicular tissue and were found to support the growth in vitro of poliomyelitis virus, Type 2, Yale-SK strain. Moreover, these cells were destroyed as a result of the multiplication of this virus within them. Similarly, "fibroblasts" propagated in primary explant cultures of testicle were destroyed by poliomyelitis viruses, Types 1 and 2. Type specific antibodies neutralized the pathogenic effect of poliomyelitis virus on monkey testicular fibroblasts. (+info)
The effect of tuberculin and cortisone singly and in combination on explanted tissues of guinea pigs, mice and rabbits.
Tuberculin (P.P.D.) and cortisone acetate, singly and in combination, were added to explanted splenic tissue derived from guinea pigs, mice, and rabbits, and buffy coats obtained from rabbits. These tissues came from normal animals or from animals which had been infected with the tubercle bacillus or immunized with killed tubercle bacilli. 5 microg./ml. of tuberculin or 0.5 microg./ml. of cortisone was the largest concentration of the respective reagents which was tolerated by cultured cells from normal animals. From the results with all tissues studied it would appear that P.P.D. selectively damaged only the growing cells of splenic tissues from sensitive guinea pigs and to a lesser degree the migrating cells of buffy coats obtained from sensitive rabbits. Cortisone appeared to have increased toxicity for only explanted splenic cells from tuberculin-sensitive guinea pigs. The specific effect of P.P.D. on tissues from tuberculin-sensitive animals was not modified by cortisone under the conditions of these experiments. Thus these data furnished no evidence that cortisone had any direct effect on the response of the sensitive cells to P.P.D. (+info)
Tissue culture studies; isolation of the active components in the ultrafilterable portion of the chick embryo extract.
A fraction of the ultrafilterable portion of chick embryo extract was isolated by alcohol extraction of a lyophilized powder of the ultrafiltrate followed by ion exchange removal of many of the inert components of the alcohol extract. This fraction contained 3 per cent of the ultrafilterable nitrogen but was capable of completely restoring the growth-promoting activity of dialyzed embryo extract, when tested with chick heart fibroblasts in roller tube cultures. The low nitrogen content, shape of the ultraviolet absorption spectrum, and presence of few free amino acids, suggest that non-dialyzable compounds serve as the chief source of nutrition for this system. (+info)
Tissue culture studies; identification of components and synthetic replacements for the active fraction of chick embryo extract ultrafiltrate.
Some of the compounds in the active fraction of ultrafiltrates of chick embryo extract have been identified as taurine, serine, glutamic acid, xanthine, uracil, glucose-6-phosphate, glucose, ferrous iron, and inorganic phosphate. Based on the identity of these compounds a synthetic replacement for the ultrafilterable portion of chick embryo extract has been devised. There is an additional nutritional requirement that can be met by vitamin B(12). Folic acid appears to be beneficial to the system though the requirements of this or any of the above compounds except vitamin B(12) remain for future research. The low nitrogen content of the isolated fraction and the synthetic mixture suggests that the main nutrition of chick cells in roller tube cultures is derived from the non-dialyzable portion of the medium. (+info)
Factors related to the growth of psittacosis virus (strain 6BC). IV. Certain amino acids, vitamins, and other substances.
The analogues of amino acids, beta-2-thienylalanine, ethionine, and 6-methyltryptophane, inhibited the growth of psittacosis virus (6BC) in tissue culture without evidence of serious toxicity for the host cells. Of a number of vitamin analogues tested, only salicyl-beta-alanide inhibited viral multiplication in the absence of toxic effects on the host cells. 6,7-Diethylriboflavin, desoxypyridoxine, and oxythiamine reduced viral growth in concentrations that possessed some toxicity for host tissue. In tolerated amounts, 3-acetylpyridine, pyridine-3-sulfonic acid, pantoyl sulfanilamide, and desthiobiotin did not effect viral multiplication. Sodium malonate inhibited psittacosis virus growth in non-toxic amounts, whereas sodium monofluoroacetate was ineffective. Colchicine suppressed multiplication of virus only after a prolonged period of exposure and subsequent delay before producing inhibition, suggesting that the effect was secondary to its antimitotic action which suppressed multiplication of the host cells. (+info)
Studies on the factors essential to the initiation and maintenance of multiplication of psittacosis virus (6BC strain) in deficient cells in tissue culture.
The growth of psittacosis virus (6BC) was studied in cultures of minced whole chick embryo tissue maintained in either Hanks-Simms solution or Hanks's balanced salt solution (BSS), and in neither medium could sustained, long-term virus growth take place. Addition of beef embryo extract (BEE) to cultures at a time when virus multiplication was declining reversed this general trend and resulted in greater virus growth. This virus-stimulating action of BEE was only partially diminished by colchicine, a mitotic inhibitor, indicating that the action of BEE was not due entirely to the development of a larger population of cells as a result of its enhancement of cell proliferation. Chick embryo tissue cultivated for 13 days in BSS prior to infection lost its ability to support the growth of psittacosis virus, but this capacity could be restored by the addition of BEE, alone or with colchicine, at the time of infection. A significant amount of virus was adsorbed to tissue in BSS alone, indicating that the failure of virus to grow in depleted tissue maintained only in BSS after infection was not due entirely to failure of virus to attach to and invade the cells. It was found that an ultrafiltrate and a dialysate of BEE contained the major part of the stimulating capacity of the whole extract, indicating that the active materials were substances of low molecular weights. Autoclaved lactalbumin hydrolysate was an active stimulator, suggesting that the materials responsible for its activity were relatively heat-stable. Since a chemically defined medium (Parker 199) was equally effective in stimulating viral growth, it should be possible eventually to define the chemical nature of the virus stimulators. The implications of the findings are discussed with special reference to their application in the study of tissue tropisms and of latency in viral infections of cells. (+info)