A STUDY OF THE MECHANISMS OF DNA AND THYMINE DEGRADATION IN CULTURED HUMAN CELLS INFECTED WITH A LIPOVIRUS

DNA and thymine degradation on cultured human, mouse, and chick cells were studied. Significant increase in DNA-degrading activity was demonstrated in human embryonic cells killed by freeze-thawing, liver cells killed with mitomycin C, mouse embryonic cells infected with encephalomyocarditis virus, and in all cells killed by the lipovirus. Twelve other viral agents, actinomycin D, and 5-fluorodeoxyuridine failed to produce a similar increase. Thymidine-2-C¹⁴-labeled cultures, either live, killed, or infected by 19 different physical-chemical and biological agents, did not release detectable quantity of C¹⁴C₂. Following infection with the lipovirus 20 to 60 per cent of the total radioactivity of thymidine-2-C¹⁴-labeled cultures was liberated as C¹⁴O₂. It was postulated that the lipovirus introduced into the host cells the missing genetic information necessary for the synthesis of one or more enzymes responsible for the reductive catabolism of thymine.     

ACTION ON FIBROBLASTS OF EXTRACTS OF HOMOLOGOUS AND HETEROLOGOUS TISSUES

Extracts of homologous adult tissues detemine an increase in the mass of pure cultures of chicken fibroblasts nourished thereon comparable to that resulting from embryonic tissue juice. But the effect of these extracts differs markedly from that of the latter, since cell multiplication does not continue indefinitely. After a few passages, the fibroblasts cultivated in adult tissue extracts grew more slowly than in Tyrode solution. The cytoplasm became dark and filled with fat granules, and death followed. It is possible that the tissues of adult animals contain, as does the serum, substances which are toxic for the homologous cells, and which progressively overcome the effect of the growth-activating substances. The effect of heterologous adult tissue extracts did not differ markedly from that of homologous tissues. The chicken connective tissue increased slightly in mass, but died sooner than the controls in Tyrode solution. By contrast, tissue juices derived from the embryos of mice, guinea pigs, and rabbits acted on chicken fibroblasts in the same manner as chick embryo juices. The increase in mass of the cultures was regular and rapid. They doubled in size every 48 or 72 hours, and the rate of growth did not decrease after 30 days. It appears that embryonic tissue juices are not necessarily toxic for heterologous fibroblasts, and that they can be used in the building up of protoplasm in the tissues of a different species. In experiments made long ago, the action of tissue juice was described as being specific. The premature death of the fibroblasts cultivated in heterologous juices at that time would now appear to have been due to spontaneous changes in the pH and the deterioration that even normal chick embryo juice at a pH of 7.8 undergoes spontaneously. In the recent experiments, when freshly prepared homologous and heterologous juices were used, their action on chicken fibroblasts in pure culture was identical. However, the fibroblasts produced in cultures nourished by rabbit juice grew better when transferred to rabbit serum than did ordinary chicken fibroblasts. It has not been determined as yet whether this effect is due to an immunization of the fibroblasts against rabbit humors, or to some decrease in the specificity eventuating in cells intermediate between rabbit and chicken fibroblasts. It may be concluded that, under the conditions of the experiments : 1. Pure cultures of chicken fibroblasts increase in mass under the influence of extracts of adult homologous tissues. But they ultimately die while the fibroblasts cultivated in embryonic tissue juices live indefinitely. 2. The increase in mass of chicken fibroblasts cultivated in the juices of mouse, guinea pig, rabbit, and chick embryos is about identical. 3. Chicken fibroblasts produced in cultures nourished by rabbit embryonic tissue juice are less sensitive to the inhibiting action of rabbit serum than ordinary chicken fibroblasts. 4. Cultures of chicken fibroblasts in extracts of adult tissues of mice, guinea pigs, and rabbits increase slightly in mass, but the increase is temporary and death occurs after a few passages.     

INHIBITION OF RELEASE OF VACCINIA VIRUS BY N1-ISONICOTINOYL-N2-3-METHYL-4-CHLOROBENZOYLHYDRAZINE

N₁-isonicotinoyl-N₂-3-methyl-4-chlorobenzoylhydrazine (IMCBH) is a selective inhibitor of vaccinia virus multiplication. In concentrations up to 50 µg/ml, IMCBH causes neither toxic morphologic changes, nor does it inhibit the multiplication of cells. Viruses other than vaccinia are not affected by IMCBH. The virus-inhibitory effect of IMCBH is dependent on the type of host cell used, i.e., the compound is effective in chick embryo fibroblasts and monkey kidney cells but not in L cells. IMCBH does not exhibit any protecting effect on vaccinia virus-infected mice or rabbits. IMCBH interferes with virus release: in single cycle experiments in chick embryo fibroblasts, IMCBH strongly blocks the release of vaccinia virus at concentrations as low as 3 µg/ml, while intracellular virus synthesis is hardly affected. Viral cytopathic changes are completely suppressed by IMCBH within the span of a single cycle infection, although extensive changes eventually occur. By inhibiting virus release from initially infected cells, IMCBH markedly inhibits the multiplication of vaccinia virus in cell cultures infected at low virus/ cell multiplicities. IMCBH does not inhibit the early toxic cytopathic changes induced by large inocula of vaccinia virus in BHK21 cells.     

STUDIES ON THE PHYSIOLOGICAL CONDITIONS PREVAILING IN TISSUE CULTURES

An analysis of some of the physiological factors active in Maitland tissue cultures has been presented in the hope that it may be of some value in clarifying the principles underlying tissue cultures in general. It has been found that the empirically determined necessity of using relatively small amounts of tissue in such cultures is dependent upon the fact that excessive tissue leads to a rapid change of reaction toward the acid side. Whereas tissue may remain viable in an environment as alkaline as pH 9 and over, viability is rapidly destroyed when the reaction approaches pH 6. Evidence is presented to indicate that the changes in electrode potentials which take place in Maitland cultures are not, as has been suggested, the determining factors upon which virus multiplication depends, although they may, of course, be incidentally important. It has been shown that there are fundamental differences between those conditions in Maitland cultures which favor the multiplication of a typical virus and those upon which the growth of the Rickettsiae of typhus fever depends. The virus which we have studied (equine encephalitis virus, western type) multiplies during the period of active tissue metabolism. The maximum virus titrations are obtained at about the time at which metabolism has come to a standstill. Thereafter the virus not only ceases to increase but rapidly deteriorates. The period of viability of the tissue cells themselves is shortened by several days in the presence of virus multiplication. There is some evidence that a temporary acceleration of oxygen uptake takes place during the time of active virus multiplication. Technical difficulties in controlling such experiments prevent certainty in regard to this point. In contrast with the conditions determining the growth of a virus agent in the Maitland cultures the multiplication of Rickettsiae does not begin to any determinable extent until after active cell metabolism has either become stabilized or has ceased. The Rickettsiae continue to grow at a time when the cells are no longer viable. It appears likely that these organisms find the most favorable conditions for growth in cells which are no longer metabolically active but in which some delicately heat-susceptible elements have not yet been disturbed. As a consequence of these observations, frozen and preserved embryonic tissues have been successfully used for Rickettsia cultivation. A report on these experiments will be made in a separate communication.     

CULTIVATION OF THE VIRUS OF GRASSERIE IN SILKWORM TISSUE CULTURES

A medium has been developed in which certain cells from the gonads of female silkworms multiply and live for periods of 2 to 3 weeks. In such tissue cultures, strains of silkworm grasserie virus were maintained in successive passages up to the number of ten. The virus multiplied greatly and typical polyhedral bodies formed in the cells of infected cultures.     

FURTHER OBSERVATIONS ON THE CULTIVATION OF VACCINE VIRUS IN LIFELESS MEDIA

We have made ten attempts to cultivate vaccine virus in tissue extracts prepared according to the method described by Eagles and Kordi (4). Renal, testicular, and chick embryo extracts were employed with a dermal strain of vaccine virus and with the Levaditi strain of neuro-vaccine virus. In no instance were we able to show that the virus multiplied in the extract media. Both of these strains of virus, however, multiplied in media containing bits of minced viable tissue. Furthermore, treatment of rabbit testicular tissue and chick embryo tissue in the manner described by Eagles and Kordi for the preparation of the extracts leaves some cells not only alive but capable of proliferation. Although the results of our work are not in accord with those obtained by Eagles and Kordi, we offer no explanation for the discrepancy. Nevertheless, one cannot examine the results of our work recorded in the six tables without recognizing the fact that in the types of media used the presence of viable cells appears to be essential for the multiplication of vaccine virus. Rabbit testicular tissue and bits of chick embryos support the regeneration of the active agent more efficiently than does rabbit renal tissue.     

Studies on persistent infections of tissue cultures. II. Nature of the resistance to vesicular stomatitis virus

Efforts were made to elucidate the nature of the resistance to vesicular stomatitis virus (VSV) observed in MCN cultures persistently infected with Newcastle disease, mumps, or 6-6 viruses (MCN_NDV, MCN_Mps and MCN_6-6, respectively). Cells derived from persistently infected cultures adsorbed VSV to the same extent as their uninfected counterparts. Only a fraction of the adsorbed virus could be recovered from the cells indicating that it enters into an eclipse in all of the cell types. While propagation of VSV in MCN cells is largely inhibited at low pH levels, the resistance of persistently infected cultures could not be ascribed to their increased lactic acid formation. Resistance was not absolute in that a few cells in persistently infected cultures apparently supported VSV reproduction. Furthermore resistance of the cultures was found to be transitory in that the VSV infection gradually gained the upper hand after 2 to 4 weeks of incubation. Addition of ultraviolet-inactivated NDV to MCN cultures induced resistance to VSV as long as the equivalent of at least one ID₅₀ (for chick embryos) of inactivated virus was provided per cell. Establishment of resistance required some time and its duration depended upon whether or not the free inactivated NDV was removed or neutralized after given adsorption periods. The transitory nature of resistance in persistently infected cultures, or in MCN cells following adsorption of inactivated NDV, is most likely explained by the fact that the cells continue to divide and that the daughter cells are, at least in part, susceptible to VSV. The results are compatible with the conclusion that the resistance observed represents another example of interference between 2 viruses.     

THE PRODUCTION OF FOREIGN BODY GIANT CELLS IN VITRO

1. Foreign body giant cells may be produced in vitro by the addition of foreign objects such as lycopodium spores and cotton fibers to cultures of chick embryo spleen. 2. These giant cells are formed by the fusion of large mononuclear wandering cells, probably endothelial cells and pulp cells. Connective tissue cells do not take part in their formation. 3. The large giant cells sometimes seen spread out over the cover-glass in cultures of chick embryo spleen are probably foreign body giant cells, the cover-glass acting as the foreign body.     

SOME CONDITIONS OF THE REPRODUCTION IN VITRO OF THE ROUS VIRUS

A method has been developed by which the susceptibility of chickens to Rous virus can be tested, and the virulence of eight or ten different fluids compared in a single animal. The results of five series of experiments made with this technique can be summarized as follows: When a medium composed chiefly of chicken serum and Tyrode solution and containing no fresh tissues is inoculated with filtered extract of Rous and other sarcomas and incubated for 48 hours, it never produces a tumor after being injected into chickens. The virus has apparently been destroyed or at least has lost activity. But, in a solid medium, composed chiefly of serum and Tyrode solution and containing fragments of fresh tissues, the virus is found to increase readily, as shown in the first series of experiments. In the course of 15 months, the experiments have been repeated many times, with identical results. Flasks containing embryo pulp or leucocytes inoculated with filtered extract of sarcoma are used to keep on hand a constant supply of the Rous virus. The cultures of monocytes inoculated with the filtered extract often assume the appearance of the cultures of Rous sarcoma. They may also remain normal to all appearance despite the circumstance that the virus is multiplying within the medium. It is not certain that the activity of Rous virus is always accompanied by cell lesions, but there is no doubt that its increase depends on the presence of fresh tissues within the medium. The disappearance of the Rous virus from a medium that does not contain any fresh tissue may be interpreted as follows: the agent has been destroyed; or it is still present in a concentration lower than 1 in 50,000, which is the concentration required to produce a tumor even in the more susceptible chickens; or, according to the hypothesis of Gye, it is present in an inactive form. In the above experiments, the fresh tissues added to the medium might conceivably have enabled the virus to keep its full activity, through supplying the conditions requisite therefor, or they might merely have furnished an activating substance. The value of Gye''s hypothesis was tested in a series of experiments. The results indicate that the tumor-producing virus present in the cultures was not composed of two parts, an inactive part multiplying in the medium, and an activating part supplied by the tissues. In another series of experiments, the relations between the reproduction of the virus and the quantity of the tissues contained in the medium were studied. The presence of a small fragment of leucocytic film or spleen tissue was sufficient to prevent the virus from disappearing. Approximately 1 c.mm. of spleen tissue in 3,000 c.mm. of medium may on occasion maintain a concentration of Rous virus in this fluid sufficient to produce a tumor upon inoculation into chickens. But this rarely happens. Generally when the medium contained only one fragment of spleen or leucocytic film, or 1 drop of embryonic pulp, the virus disappeared rapidly. When the quantity of tissue was from five to nine times larger, an abundant production of virus was practically always found. It became obvious that the quantity of active virus present in a medium containing multiplying cells depends upon the amount of tissue in the medium. In the fourth series of experiments, the kind of cells needed for the multiplication of the virus was ascertained. Rous virus was found to disappear rapidly from the fluid of cultures of fibroblasts, while it multiplied readily in cultures of leucocytes, the total volume of both tissues being approximately the same. It should be remembered that strains of fibroblasts obtained from Rous and other sarcomas very rarely produce tumors upon inoculation into chickens, while the inoculation of cultures of macrophages from the same tumors practically always determines their appearance. The fifth series of experiments showed that the cell metabolism is an important factor in the reproduction of the virus. When the activity of tissues had been suppressed or very much decreased by freezing, no virus was produced, while it multiplied readily in the control. The lack of oxygen for a period of 24 or 48 hours stopped cell proliferation, and at the same time the production of Rous virus ceased. However, the fluid of some of these dead or inactive cultures, after 6 days incubation, was still able to give rise to a small tumor upon inoculation into a chicken. There is an evident relation between the proliferating activity of the tissues and the production of the virus. But the agent may persist for several days in association with dead tissues. It may be concluded that the reproduction in vitro of the active virus depends on the presence of fresh tissues in the culture and upon the quantity, the activity, and the nature of the cells contained in the medium.     

STUDIES ON THE ETIOLOGY OF PRIMARY ATYPICAL PNEUMONIA : II. PROPERTIES OF THE VIRUS ISOLATED AND PROPAGATED IN CHICK EMBRYOS

Experiments to determine the optimum conditions for propagation of the virus of atypical pneumonia in chick embryos are described. Variations in the activity of infected chick embryo material were investigated. The highest dilution of chick embryo suspension producing pulmonary lesions in hamsters and cotton rats is not over 10^–3. Dilutions of 10^–4 infect chick embryos. The virus is unstable at room temperature and also loses activity when stored in a dry-ice refrigerator unless the suspensions are kept in sealed glass tubes. Filtration experiments indicate a maximum particle size of 180 to 250 mµ. The virus propagated in chick embryos produces pulmonary lesions in hamsters and cotton rats which have been immunized to their own non-bacterial agents inducing pulmonary lesions. Of these, the pneumonia virus of hamsters most frequently causes intercurrent respiratory infections, and methods of controlling epizootics due to this agent are described.     

ENUMERATION OF CELL-INFECTING PARTICLES OF NEWCASTLE DISEASE VIRUS BY THE FLUORESCENT ANTIBODY TECHNIQUE

A procedure has been developed for the determination of the concentration of infective Newcastle disease virus (NDV) based on the enumeration of singly infected and distributed HeLa cells which are visualized by staining with fluorescent antibody. Infective virus assayed by the fluorescent cell-counting procedure is expressed in terms of cell-infecting units (CIU). Adsorption of NDV to HeLa cell monolayers reached a plateau 1 to 1.5 hours after inoculation of coverslip cultures, and 12 per cent of the infective particles inoculated failed to adsorb. The half-life of NDV in protein-free Eagle's medium at 37°C. was 2.1 hours. There was a linear relationship between virus concentration and the number of infected cells. The coefficient of variation of the mean of replicate determinations of infective NDV was 8.2 per cent. The distribution of single infected HeLa cells in the monolayer corresponded to the Poisson distribution. With NDV the cell-infecting unit (CIU) determined in HeLa cells is equivalent to the plaque-forming unit in chick embryo cells and the egg infective dose. In experiments on the mechanism of dissemination of NDV in monolayer cultures of HeLa cells, NDV was found to spread from cell to cell through the extracellular milieu.     

INCOMPLETE SIMIAN PAPOVAVIRUS SV40 : FORMATION OF NON-INFECTIOUS VIRAL ANTIGEN IN THE PRESENCE OF FLUOROURACIL

A study was made of the effects of 5-fluorouracil (FU) and 5-fluorodeoxyuridine (FUDR) on the replication of the simian papovavirus SV40 in cercopithecus monkey kidney cells and on the production of virus antigen by these cells. Both drugs markedly suppressed the production of new infectious virus by SV40-infected cells. Synthesis of viral protein was also markedly suppressed by FUDR, but not by FU. In the presence of FU, infected cells produced large amounts of viral protein which were detected by the fluorescent antibody technique. The antigen was not distributed in a particulate fashion as in untreated cells. Diffuse virus antigen was observed in the nuclei of FU-treated cells, resembling the distribution of antigen near the end of the eclipse period in untreated, infected cultures. This stage of antigen production presumably preceded viral assembly. Virus particles with or without cores were rarely seen with the electron microscope in infected FU-treated cells, although large numbers of SV40 particles were readily visualized in untreated, infected cells. It appears that at least one antigenic protein of this papovavirus is synthesized abundantly in FU-treated cells, but is not assembled into virus shells in the presence of the inhibitor.     

FACTORS OF RESISTANCE TO HETEROPLASTIC TISSUE-GRAFTING : STUDIES IN TISSUE SPECIFICITY. III.

It has been shown that the chick embryo offers suitable conditions for the growth of implanted tissues, whether these be embryonic or adult, of the same species or a foreign one. The chick at about the time of hatching develops a defensive mechanism against the tissue of foreign species. This resistance can be supplied to the embryo in the early stages if grafts of adult spleen or bone marrow are implanted. Under these conditions the embryo exhibits the same resistance to foreign tissue as does the adult, and presents the same histological manifestations about the graft. Furthermore, the same tissues, spleen and bone marrow, when grafted into an embryo with an established and growing rat tumor, bring about a retrogression and absorption of the foreign tissue. Other adult tissues do not supply this power to the embryo.     

DEPRESSION OF ANAEROBIC GLYCOLYSIS OF EMBRYONIC TISSUE BY WESTERN STRAIN OF EQUINE ENCEPHALOMYELITIS VIRUS. PREVENTION OF THIS EFFECT BY SPECIFIC IMMUNE SERUM

Studies were made on the effect of mixing the Western strain of equine encephalomyelitis virus (W.E.E.) and embryonic tissue on the rate of anaerobic glycolysis of the tissue. Whole chick embryo, chick embryo from which brain and spinal cord had been removed, and embryonic skeletal muscle were employed. 1. W.E.E. virus depressed the rate of anaerobic glycolysis of embryonic tissues within 2 days after its addition to the tissue. The decrease in anaerobic glycolysis varied from 17 to 82 per cent and was apparent 2 to 4 days after the addition of the virus. No significant effect of the virus was observed 4 hours and 6 days after mixing it with the tissue. 2. Anti-W.E.E. immune serum prevented the inhibiting action of W.E.E. virus on the anaerobic glycolysis of embryonic skeletal muscle.     

TYPHUS FEVER : IV. FURTHER OBSERVATIONS ON THE BEHAVIOR OF RICKETTSIA PROWAZEKI IN TISSUE CULTURES

In tissue cultures grown at 32°C., typhus Rickettsiae increase rapidly within the cytoplasm of infected cells up to about the 14th day. At this time practically every cell is infected and the majority of cells are distended with organisms. This condition remains constant as long as successful cultures of the cells can be maintained (up to 52 days). Loss in virulence does not take place during this period in vitro. The number of Rickettsia-filled cells found in sections and the incubation period of the infection resulting from inoculation of cultures from each age group are definitely correlated. The behavior of typhus Rickettsiae in dividing cells is described and methods of spread of the infection other than by mitosis of cells are discussed. Normal tissues do not become infected in vitroto any considerable extent in spite of prolonged proximity to heavily infected cultures of scrotal sac exudate. Complete anaerobiosis and alterations in pH do not alter the intracellular location of the organism in tissue cultures. The organisms are not seen within nuclei of infected cells. They remain intact and infective for several weeks in cells which are kept alive but not multiplying. They disappear in less than 1 week, however, when the cells undergo degeneration.     

THE SUSCEPTIBILITY OF CHICK EMBRYO SKIN ORGAN CULTURES TO INFLUENZA VIRUS FOLLOWING EXCESS VITAMIN A

The conversion of chick embryonic epidermis to mucous epithelium by excess vitamin A in organ culture as reported by Fell and Mellanby (5) was shown to be accompanied by a corresponding change of susceptibility to influenza and vaccinia viruses. Untreated epidermis of 10- to 12-day chick embryos supported the growth of influenza (PR8) virus in organ cultures and a maximum infectivity (EID₅₀) titer was reached 2 to 3 days after infection. At the same time) the epidermis showed squamous keratinization, beginning about the 4th day of cultivation. Addition of excess vitamin A (40 µg per ml) to the skin organ culture induced the following changes: (a) mucous metaplasia of the epidermis which was usually first evident after 4 to 5 days in the vitamin A medium, (b) increase in the daily and maximum yield of influenza virus, if the epidermis had been grown for 4 or more days in the vitamin A medium before infection took place, and (c) decrease in the production of vaccinia virus under similar conditions. The maximum yield of both viruses remained unchanged, however, if excess vitamin A was introduced to the organ culture at the time of virus inoculation. The magnitude of increase in the yield of influenza virus in this organ culture system was found to be proportionally related to the concentration of vitamin A added 4 or more days before inoculation of this virus. Increasing doses of vitamin A however, had no effect on the short-term growth of influenza virus in tissue cultures of chorio-allantoic membrane. Observation on the early period (2 to 12 hours) of influenza virus growth initiated in the 4-day organ cultures of chick embryonic skin showed no significant difference in virus production between the normal and the vitamin A medium groups. The change of virus specificity apparently is not due to the presence of excess vitamin A per se, but appears to be related to the change of differentiation produced in the organ culture system.     

STUDIES UPON THE CHARACTERISTICS OF DIFFERENT CULTURE MEDIA AND THEIR INFLUENCE UPON THE GROWTH OF TISSUE OUTSIDE OF THE ORGANISM

1. There is a great difference between embryonic and adult tissue as far as their growth outside of the organism is concerned. Adult tissue grows only in plasma. Embryonic tissue grows also very well in serum and serum plus agar. In Ringer''s solution and in Ringer''s solution plus agar no growth occurs, whether embryonic or adult tissue is employed; survival and emigration of cells are seen to some extent. 2. For the growth of connective tissue cells of chick embryo, unheated homogenic serum is a better culture medium than heated serum. The growth of epithelial cells is not thus influenced. 3. Heated heterogenic serum is a better culture medium for growth of embryonic connective tissue cells than unheated. 4. There is an inverse ratio between the hemolytic power of heterogenic sera and the extent of growth of tissue in them. This inverse ratio is not found in heterogenic plasmas.     

ELEMENTARY BODIES OF VACCINIA FROM INFECTED CHORIO-ALLANTOIC MEMBRANES OF DEVELOPING CHICK EMBRYOS

By a method of differential centrifugation and tryptic digestion suspensions of elementary bodies have been prepared from chorioallantoic membranes of chick embryos infected with vaccine virus. The infective titer of the final suspension of elementary bodies was usually the same as that of the original tissue emulsion. Elementary bodies from infected chick membranes were agglutinated as well by antivaccinal serum obtained from different mammalian species as were bodies prepared from inoculated rabbit skin. Seitz filtrates of infected chick material contained soluble precipitable substances of vaccinia; these filtrates and filtrates from infected rabbit skin, respectively, reacted equally well with rabbit serum which contained either L or S antibodies.     

STUDIES ON THE CULTIVATION OF THE VIRUS OF LYMPHOGRANULOMA VENEREUM

Two strains of the lymphogranuloma venereum virus were maintained in tissue cultures for 12 and 21 generations respectively. In a third strain a quantitative increase in potency as high as 1000 times was obtained by inoculating tissue cultures with infected emulsions of known virulence. Greater increases in potency were consistently obtained by maintaining tissue cultures and virus at room temperature (23°C. with a range of approximately ± 5°C.) than by incubating them at 37°C. The virus did not survive in the absence of oxygen. Embryonic guinea pig brain and serum ultrafiltrate were found to be the most effective vehicles for propagation of the lymphogranuloma virus. There is evidence that the site of activity for this virus is intracellular. Embryonic guinea pig brain cells were maintained in the serum ultrafiltrate diluted with buffered salt solution in good (morphologic) condition for as long as 70 days. Not only could old cultures be successfully inoculated with the virus of lymphogranuloma, but high titres could be maintained over extended periods.     

INTERFERENCE BETWEEN VIRUSES IN TISSUE CULTURE

The influence of one virus on the growth of another in tissue culture was investigated. The 17DD High strain of yellow fever virus was found capable of completely suppressing the growth of both the Asibi strain of the same virus and of the heterologous West Nile virus, even when these were added to the cultures in large amounts. The 17DD High strain of yellow fever virus and the West Nile virus produced either partial or complete suppression of growth of the Venezuelan equine encephalomyelitis virus, depending upon the quantity of the latter inoculated into the cultures. Owing to lack of methods for the detection of interference except in a single direction, reciprocal interference with these viruses could not be investigated. The 17DD High strain of yellow fever virus and the West Nile virus were able to suppress completely, or almost completely, the growth of influenza A virus added to the infected cultures in maximal amounts. Interference in the reverse direction, even with the use of small amounts of the neurotropic viruses, was not demonstrable. Cultures infected with the 17DD High strain of yellow fever virus were examined for the presence of neutralizing antibodies and non-specific antiviral substances; neither was found present.