PROPAGATION OF RABIES VIRUS IN TISSUE CULTURE

Rabies virus has been propagated in serum-Tyrode solution containing either embryo mouse brain or embryo chick brain. The culture virus reached a titre of 3 x 10^–5 cc. after 4 days'' incubation at 37°C., and survived at least 2 months at 5°C. in the liquid or dry state.     

THE ROLE OF INTERFERON IN VACCINIA VIRUS INFECTION OF MOUSE EMBRYO TISSUE CULTURE

The production of an interferon-like substance by vaccinia virus is described. The physical properties of vaccinia interferon are shown to be similar to those of previously reported interferons. Data defining the role of vaccinia interferon in cell resistance and in establishment and maintenance of a carrier culture are presented. Elimination of virus from an infected culture is demonstrated and the role of interferon in the recovery process is considered.     

DISEASE ACCOMPANYING IN UTERO VIRAL INFECTION : THE ROLE OF MATERNAL ANTIBODY IN TISSUE INJURY AFTER TRANSPLACENTAL INFECTION WITH LYMPHOCYTIC CHORIOMENINGITIS VIRUS

Early, after in utero infection with LCM virus, SWR/J and HA/ICR mice developed manifestations of immune complex disease. Observations based on nursing such mice with virus-infected, immune, or noninfected mouse mothers indicated that maternal antiviral antibody was responsible for the early immune complex glomerulonephritis. Despite comparable viral persistance, in utero-infected offspring failed to develop glomerulonephritis when nursed by noninfected mouse mothers, but did when suckled by virus-infected mouse mothers. Nursing by mouse mothers carrying high titers of anti-LCM viral antibody markedly enhanced the Ig glomerular deposits and the resultant nephritis.     

STUDIES ON GONOCOCCUS INFECTION : IV. PILI: THEIR ROLE IN ATTACHMENT OF GONOCOCCI TO TISSUE CULTURE CELLS

Attachment of Neisseria gonorrhoeae to amnion cells in tissue culture is facilitated if the gonococci bear pili. This has been determined by studying the number of pilated, colony type 2 gonococci associated with amnion cells after incubation in vitro as compared with the number of nonpilated, colony type 4 gonococci present with amnion cells under the same conditions. These data are supported by light microscope findings. Electron microscope studies provide visualization of fine structure of gonococcal attachment. Gonococci are also found within amnion cells in this in vitro system.     

STUDIES ON THE PROPAGATION IN VITRO OF POLIOMYELITIS VIRUSES : I. VIRAL MULTIPLICATION 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 10¹⁵ to 10^95.3. The LD₅₀ 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.     

ENDOSYMBIOTIC RELATIONSHIP BETWEEN ANIMAL VIRUSES AND HOST CELLS : A STUDY OF RABIES VIRUS IN TISSUE CULTURE

RE cultures infected with a fixed rabies virus were studied. The virus can propagate itself in these cells for an indefinite period of time without interfering with cell growth. The present study characterizes this truly endosymbiotic relationship. Virus-specific antigen was detected in the cytoplasm of each cell by fluorescein-labeled antirabies serum but only 4 to 5 per cent of the cells released infectious virus. All cells undergoing division showed viral antigen throughout the mitotic process. Also, the growth rate, plating efficiency, and morphological characteristics of both the infected and control cultures were identical. No difference was detected between the RE and the RE-CVS cell populations by RNA and DNA labeling experiments, using H₃-thymidine, and H₃-uridine. Although antirabies serum effectively inhibited the spread of extracellular virus, it did not interfere with cell-to-cell transmission of the virus during mitosis, in the course of 9 cell transfers during 53 days. RE-CVS cells, when exposed to fresh antirabies serum, lysed completely but inactivated serum had no lytic effect. The addition of fresh hamster complement to the inactivated serum restored its cytolytic properties. The serially passaged RE-CVS virus gradually became less virulent for mice and displayed a weak antigenicity in the mouse protection test. Another feature of the RE-CVS cell system is its resistance to infection with homologous and heterologous viruses despite the apparent absence of an interferon-like substance.     

ANTIBODY PRODUCTION BY CELLS IN TISSUE CULTURE : I. MORPHOLOGICAL EVOLUTION OF LYMPH NODE AND SPLEEN CELLS IN CULTURE

An organ culture technique was used to investigate the migration and the morphological evolution of lymphocytes from lymphopoietic tissues. This evolution was compared with the behavior of cells extracted from the tissue and kept in nutritive medium in vitro. It was found that cells were continuously migrating from the fragments of lymph nodes or spleen, and were attaching to the glass. They spread on glass, their protoplasm enlarged and their nucleus became clearer. The evolution towards blastoid cells was identical with that described under artificial stimulation by PHA for example. Cytological identification of the cells actively engaged in antibody synthesis (as detected by local hemolysis in gum) at the time of staining, showed that several distinct cellular types were active, including plasma cells and macrophagelike cells. It is assumed that the stimulated lymphocytes, after spontaneous migration from the tissue are able to evolve into an "immunoblast" stage and then, eventually after fixation upon a physical support, to initiate antibody synthesis.     

INDUCTION OF ARGINASE ACTIVITY WITH THE SHOPE PAPILLOMA VIRUS IN TISSUE CULTURE CELLS FROM AN ARGININEMIC PATIENT

Inoculation of the Shope virus in tissue cultures of human fibroblasts from a patient with a deficiency of the enzyme arginase results in an induction of arginase activity, apparently virus coded.     

STUDIES ON PERSISTENT INFECTIONS OF TISSUE CULTURES : V. THE INITIAL STAGES OF INFECTION OF L(MCN) CELLS BY NEWCASTLE DISEASE VIRUS

The initial stages of infection of L(MCN) cell populations with standard Newcastle disease virus (NDV_ST) were analyzed in an effort to elucidate the steps leading to survival of the cultures and to the indefinite persistence of the infectious process at a low level. Cells were exposed in suspension to NDV at varying multiplicities and the monolayer cultures derived from such cells assayed at intervals for cellular growth rates, percentage of infected cells as determined by immunofluorescence, yields of viral progeny and of interferon, and, on occasion, resistance to superinfection with vesicular stomatitis virus. The percentage of cells calculated to be initially infected on the basis of adsorption data was found to match closely the percentage of immunofluorescent cells resulting from the first infectious cycle (up to 24 hours). Cells initially infected with NDV_ST produced a mixed progeny of infectious virus (from 15 to 40 pfu/cell) and about 10 times as many non-infectious particles in 24 hours [NDV_L(MCN)], but little or no interferon. If all cells were infected the cultures ultimately died. At multiplicities of infection (m) of 2 or less the cultures survived with increasing ease as the percentage of infected cells was reduced. The number of pfu per infected cell was of the above order during the first 3 days; it declined thereafter. Limited secondary spread of the infection was noted by 48 hours and no further cycling was noted thereafter. As m decreased from 2.0 to 0.1 there was an increase in the yields of interferon and the time at which peak titers were reached. Addition of anti-NDV serum 2 hours after infection prevented measurable production of interferon. In contrast, following exposure of cells to NDV_L(MCN) at multiplicities ranging from 20.0 to 0.2 (based on infectious virus) all cultures survived, no secondary spread was noted, the number of pfu per infected cells was reduced at the higher multiplicities, and the yields of interferon were similar and maximal by 24 hours and not affected by anti-NDV serum added after an adsorption period of 2 hours. It is concluded that the non-infectious virus particles in the progeny released from NDV_ST-infected cells induce resistance in remaining cells or, if adsorbed simultaneously with infectious virus, abort the intracellular infectious process. In both instances interferon is produced which may then render additional cells resistant. The non-infectious component is considered an incomplete or defective product of viral replication and not merely thermally inactivated virus. NDV_ST partially or completely inactivated at 37°C induced neither cellular resistance nor synthesis of interferon. The incomplete viral component behaved in all respects like ultraviolet-inactivated NDV_ST except that it was significantly more efficient in inducing interferon synthesis. On the basis of the presented data a scheme has been devised and discussed which appears to explain satisfactorily the events which take place on initial infection of L(MCN) cells with NDV and which lead to the persistence of the infectious process.     

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.     

PATHOGENESIS OF H-1 VIRUS INFECTION OF EMBRYONIC HAMSTER BONE IN ORGAN CULTURE

H-1 virus infection of hamsters has been shown to produce runting, microcephaly, cranial lacunae, and deformed teeth in animals inoculated during the suckling period and to cause various abnormalities, including skeletal defects, in embryos infected transplacentally. To explore the pathogenesis of these effects of viral infection on bone, the response of embryonic hamster tibiae in organ culture to inoculation with the H-1 strain of picodna virus was studied. This system made possible the direct observation of the reaction of bone to virus in a regulated environment. During a period of 7–17 days after inoculation the following observations were made: (a) H-1 virus was found to infect and replicate in bone. (b) Infected bones became more translucent, slender, and elongated than control bones. (c) Bone growth as measured by increase in wet weight was reduced in infected tibiae. (d) Infected bones showed periosteal and perichondral degeneration and diminished deposits of subperiosteal bone. It was concluded that the skeletal abnormalities which develop in embryonic and suckling hamsters after H-1 virus inoculation are the direct result of viral replication in bone, and that indirect phenomena such as those associated with chronic infection need not be postulated to explain the deformities seen in these animals.     

STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) : II. IMMUNOLOGICAL EVIDENCE OF LATENT INFECTION WITH THE VIRUS IN NUMEROUS MAMMALIAN SPECIES

The results of neutralization tests with PVM and serum obtained from numerous animal species indicate that antibodies agaiust this virus were present in the blood of all mammalian species tested, as not in that of fowls, and that their incidence in various species was widely different. They indicate, also, that in certain species, particularly the cotton rat, there were marked seasonal variations in the incidence of such antibodies; in the late winter and spring the incidence was much higher than during the summer and fall seasons. Cotton rats and hamsters which did not possess neutralizing antibodies against PVM were susceptible to manifest pulmonary infection with this virus, irrespective of the effects of previous experiments upon them, whereas those which possessed such antibodies were immune. It is suggested that circulating antibodies against PVM were present as a result of preceding infection with a latent virus; either PVM or an agent closely related to it in antigenic composition. Appropriate non-specific stimuli, e.g. the intranasal injection of suspensions of normal chick embryos, induced the development of neutralizing antibodies against PVM with significantly greater frequency in each of three species than occurred in control animals. Materials derived from patients with primary atypical pneumonia yielded results almost identical to those obtained with normal chick embryo suspensions. It is suggested that such materials, like the other non-specific stimuli employed, were effective in evoking a specific antibody response, because they unbalanced an equilibrium which previously existed between animal host and latent pneumotropic virus.     

FACTORS MODIFYING HOST RESISTANCE TO VIRAL INFECTION : III. EFFECT OF WHOLE BODY X-IRRADIATION ON EXPERIMENTAL ENCEPHALOMYOCARDITIS VIRUS INFECTION IN MICE

The resistance of mice to encephalomyocarditis (EMC) virus was markedly decreased by prior exposure to whole body X-irradiation. In contrast to non-irradiated controls, the course of EMC virus infection in X-irradiated animals was characterized by (a) an enhanced mortality, (b) shortening of the incubation period, (c) higher levels of virus in the blood during the viremic phase and persistence of the viremia until death, (d) failure to develop detectable serum levels of neutralizing antibody, and (e) the earlier appearance and higher levels of virus in brain and heart tissue. The level of interferon in the serum during the course of infection was similar in both groups. The administration of relatively small quantities of anti-EMC virus neutralizing antibody to X-irradiated mice during the early phases of the infection with EMC virus restored their resistance to levels comparable to nonirradiated animals. An alteration of local organ defense mechanisms in the central nervous system could not be demonstrated. It is proposed that (a) the inability of the X-irradiated animal to elaborate specific neutralizing antibody was a critical determinant in their failure to clear the viremia, (b) this increase in the level and duration of the viremic phase resulted in the exposure of target organs to a greater inoculum of virus, and (c) the enhanced mortality observed in irradiated mice reflected this greater target organ involvement. The experimental model presented, therefore, suggests that the immunologic response is a critical determinant of host resistance during this primary systemic virus infection.     

THE INFLUENCE OF THE PARTNER CELL ON THE PRODUCTION OF L VIRUS AND THE EXPRESSION OF VIRAL SURFACE ANTIGEN IN HYBRID CELLS

The C-type particles produced by the A9 and A9HT sublines of mouse L cells were shown to infect C3H (N type), but not C57BL (B type), mouse embryo fibroblasts. Infection was indicated by distinct single giant cell formation in the XC monolayer used to overlay the mouse embryo fibroblasts. On the basis of these results it was concluded that the L cell virus is N tropic. A9 and A9HT cells were fused to various mouse cells derived from tumors and normal tissues. The ability to produce the Moloney-type surface antigen and to release infectious virus was introduced by the A9 component into the hybrid cell. Virus production, measured by antigen induction on JLS-V9 cells, was suppressed in those hybrids in which the partner cell had a genotype determining low infectibility with that particular virus (B-type cell). It thus appears that the major genetic locus affecting resistance to infection with leukemia viruses, the Fv-1 locus, regulates infectious virus production in somatic cell hybrids also. The same genetic locus did not seem to govern the expression of all virus-related functions, for the virus-determined membrane antigen was demonstrated in many of the N x B-type hybrids in which production of infectious virus was suppressed.     

THE TOXICITY OF STREPTOLYSIN O FOR BEATING MAMMALIAN HEART CELLS IN TISSUE CULTURE

Pulsating mammalian myocardial cells were found to be highly susceptible in tissue culture to rapid destruction by streptolysin O. Cessation of beating occurred almost immediately, followed within minutes by multiple cell membrane bleb formation. Parallel with these changes, the cytoplasm became intensely granular and the nuclear membrane apparently thickened when viewed by phase microscopy. At the ultrastructural level, the cell membrane blebs were found to contain relatively small numbers of granular fragments. The endoplasmic reticulum of damaged heart cells was quite swollen, and its contents were considerably condensed. The myofibers were not strikingly altered, but cytoplasmic and mitochondria vacuoles were rather abundant. Cardiac endothelial, kidney epithelial, and fibroblast cells were also susceptible to lysis by this toxin, but the reactions occurred more slowly or bleb formation was less evident. An antiserotonin drug known to be protective against streptolysin-O in vivo (UML-491), did not protect against killing of cardiac cells at the tissue culture level. Serotonin could not be detected in the culture fluid after lysis of cardiac cells by streptolysin O.     

THE INFLUENCE OF CORTISONE ON EXPERIMENTAL VIRAL INFECTION : VII. KINETICS OF INTERFERON FORMATION AND ITS INHIBITION WITH HYDROCORTISONE IN RELATION TO VIRAL STRAIN AND VIRULENCE

A comparative study was undertaken of the pathogenesis of infection of the allantoic sac of the chick embryo with three influenza viruses of differing virulence, and of the influence of hydrocortisone on the course of infection. Judged on the basis of earlier onset and greater degree of inflammatory response and diminished survival time of infected embryos, Mel. and Lee viruses were markedly more virulent than PR8, despite the earlier appearance of virus in PR8-infected embryos. Interferon appeared first and in greater quantity in the allantoic fluid of Lee-infected embryos and latest with PR8 infection. Thus, there was no correlation of avirulence and better interferon production with the viruses under study in the present system. Furthermore, evidence obtained suggested that Lee virus ("virulent") was most susceptible to interferon action, and also that viral synthesis in the chorioallantoic membrane with PR8 ("avirulent") persisted after the appearance of interferon. The injection of hydrocortisone within 2 hr of the initiation of infection delayed the synthesis of all three viruses; had no significant effect upon the inflammatory response; and transiently inhibited the synthesis of interferon, while prolonging the survival of Lee- and Mel.-infected embryos. Late administration of hydrocortisone suppresses both the inflammatory response and the production of interferon. Only in the case of Lee virus infection did hydrocortisone administration lead to augmentation of final yields of virus with the low infection multiplicity employed in the present experiments. It is postulated that Lee virus is a better inducer of interferon because its infectivity in vivo is more rapidly inactivated. As a consequence synthesis of Lee virus is more under the control of endogenous interferon than is the case with PR8 or Mel. virus. Therefore, inhibition of interferon synthesis with hydrocortisone has a greater influence on final yields of Lee virus.     

STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) IN CELL CULTURE : I. REPLICATION IN BABY HAMSTER KIDNEY CELLS AND PROPERTIES OF THE VIRUS

Pneumonia virus of mice (PVM) has been serially propagated in a line of baby hamster kidney (BHK21) cells. A maximum titer of 6.3 x 10⁶ TCID₅₀ per ml was obtained, and there was little variation in yield on serial passage. PVM grown in BHK21 cells was antigenically similar to virus obtained from the mouse lung, but was somewhat less virulent for the mouse after 10 serial passages in these cells. Virus produced by BHK21 cells agglutinated mouse erythrocytes without prior heating or other treatment. Sedimentation of PVM in the ultracentrifuge or precipitation by ammonium sulfate resulted in a loss in infectivity but an increase in hemagglutinating activity, presumably due to disruption of the virus particle. In a potassium tartrate density gradient, the major portion of infective virus sedimented at a density of approximately 1.15, and noninfective hemagglutinin, at a density of approximately 1.13. Stock virus preparations appear to contain a large amount of noninfective hemagglutinin. The replication of PVM was not inhibited by 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. Infected cells contained eosinophilic cytoplasmic inclusions which showed the acridine orange staining characteristic of single-stranded RNA. Foci of viral antigen were observed in the cytoplasm of infected cells by fluorescent antibody staining. The results suggest that PVM is an RNA virus that replicates in the cytoplasm.     

THE PROPAGATION OF THE VIRUS OF EPIZOOTIC HEMORRHAGIC DISEASE OF DEER IN NEWBORN MICE AND HELA CELLS

The New Jersey strain of EHD virus has been propagated in newborn Swiss mice by the intracerebral route and is regularly lethal beyond the first serial mouse passage. A complement-fixing antigen prepared from the brains of infected mice reacts positively with the sera of deer recovered from infection with either the New Jersey or South Dakota strain of virus, but not with the serum of normal deer. The mouse-passaged virus induced an inapparent infection in an experimental deer. The virus can also be grown serially in HeLa cell culture and induces a characteristic cytopathic effect. It is neutralizable in such cultures to high titer by the sera of deer recovered from EHD (New Jersey strain) and to lower titer by the serum of a deer recovered from EHD (South Dakota strain). Normal deer serum does not neutralize the virus in tissue culture. The HeLa cell-passaged virus induced typical lethal EHD in an experimental deer and virus could be recovered from most of the tissues of this animal in HeLa cell culture. An unexplained prozone of inhibition of cytopathogenicity at low dilutions was observed in cultures of some of the organs. The fact that EHD virus exhibited a limited sensitivity to sodium desoxycholate suggests that it may belong in the arbor virus group.     

THE ENDURING PARTNERSHIP OF A NEOPLASTIC VIRUS AND CARCINOMA CELLS : CONTINUED INCREASE OF VIRUS IN THE V2 CARCINOMA DURING PROPAGATION IN VIRUS-IMMUNE HOSTS

The V2 carcinoma—a transplanted rabbit cancer derived originally from a virus-induced papilloma and carrying in masked or altered form the virus primarily responsible for it—was propagated in five successive groups of animals all previously hyperimmunized against the papilloma virus. The cancer grew as well in the hyperimmunized hosts as in normal animals implanted during the same months; and serological tests, made when the tumor was eventually returned to ordinary hosts, proved that the virus was still associated with the carcinoma cells: it had increased to the usual extent as the tumor grew in the hyperimmune animals. The continued increase of the neoplastic virus during propagation of the V2 carcinoma in hyperimmunized hosts contrasts sharply with the elimination of certain extraneous passenger viruses when the tumors they ride upon are grown in hosts previously immunized against them. The facts as a whole would seem to warrant a distinction between the enduring partnership of a neoplastic virus and carcinoma cells on the one hand and the casual association of passenger viruses with tumor cells on the other.     

ANTIBODY PRODUCTION BY CELLS IN TISSUE CULTURE : II. QUALITATIVE AND QUANTITATIVE ASPECTS OF ANTIBODY PRODUCTION (LOCAL HEMOLYSIS IN GUM) BY CELLS OBTAINED FROM LONG TERM CULTURE

By combining a tissue culture method with the detection of antibody-producing cells by local hemolysis in gum it has been possible to follow the immunological activity of cells from tissue fragments for long period of time. These fragments were obtained from lymph nodes or spleens of rabbits immunized by sheep erythrocytes. It was found that, while the immunological activity of the free cells in suspensions decreased fast and disappeared in a few days, the cells attaching on glass could express their activity for at least 3 wk. It is assumed that these cells are the daughters of cells from the fragments which were not active antibody producers at the beginning, but differentiated, during the culture, into cells endowed with two capacities: glass adherence and antibody synthesis. One can further admit that the type of culture employed exerts a selective pressure favoring formation of antibody-producing cells.