Latent viral infection of cells in tissue culture. IV. Latent infection of L cells with psittacosis virus

By maintaining L cells in a balanced salt solution of inorganic salts and glucose (BSS) for 2 days or more, they are rendered incapable of supporting the growth of psittacosis virus (6BC), though it infects such cells and is present intracellularly for as long as 3 days in a non-infectious phase. The addition of an enriched medium to such a culture of cells at any time up to 4 days after infection results in the appearance of infectious virus within these cells, which multiplies and is released from the cells, providing the entire period of exposure of such cells to the BSS does not exceed 6 days, following which the cells die. A latent infection with psittacosis virus in a non-infectious phase has been established in a pure line of cells which possess properties of malignancy.     

Latent viral infection of cells in tissue culture. II. Relationship of cell nutrition to initiation of growth of psittacosis virus

When chick embryo tissues cultivated for 13 days in Hanks's balanced salt solution (BSS) were infected with psittacosis virus (6BC), they did not support active viral multiplication until synthetic medium 199 of Parker (3) was added. By testing various combinations of the substances in this and other synthetic media, it was found that the minimum number of compounds required to effectively stimulate virus growth in the presence of BSS comprised the amino acids and water-soluble vitamins found in medium 199. Addition of either amino acids or water-soluble vitamins alone to BSS resulted in only slight stimulation of viral proliferation. Many constituents of the synthetic media were found not to be essential to the stimulation of viral multiplication. The following substances added to a medium containing amino acids and water-soluble vitamins in BSS failed to increase the quantity of virus produced: diphosphopyridine nucleotide (DPN), triphosphopyridine nucleotide (TPN), coenzyme A, the fat-soluble vitamins, ribose sugars, and three biological reducing agents: cysteine, glutathione, and ascorbic acid. Among other substances that proved to be not essential a group of purines and pyrimidines present in medium 199 were found to be probably toxic to cells in the concentrations used, since virus titers were lower in media containing these compounds than in those from which they were absent. A change in the nutritional status of these cells involving amino acids and water-soluble vitamins has thus permitted to transform a latent, undetectable viral infection to an inactive infection in vitro.     

Latent viral infection of cells in tissue culture. VI. Role of amino acids,glutamine, and glucose in psittacosis virus propagation in L cells

Mouse fibroblasts (L cells) fail to support the growth of psittacosis virus (6BC strain) if they are maintained on a medium containing only inorganic salts and glucose for 2 days prior to infection. Virus propagation can be stimulated by the addition of a synthetic medium containing amino acids, water-soluble vitamins, glutamine, glucose, and inorganic salts. By omitting single amino acids from the complete synthetic medium, tyrosine, threonine, methionine, isoleucine, phenylalanine, tryptophan, leucine, valine, and cysteine or cystine were found to be essential for stimulation, while lysine, arginine, histidine, hydroxyproline, proline, glutamic acid, aspartic acid, serine, alanine, and glycine were not essential. The cells on deficient media showed varying degrees of degenerative changes, but there was little correlation between ability to support psittacosis virus growth and morphologic condition of the cells. Glucose is also an essential component of the medium for viral growth, but the absence of glutamine had no effect on stimulation of virus propagation. L cell cultures maintained on media deficient in phenylalanine or tryptophan for 2 days before infection were also found to be incapable of supporting virus growth. The implications of this study in latent viral infections are discussed.     

Studies of influenza virus infection in the chick embryo using fluorescent antibody

As evidenced by specific staining with fluorescent antibody, the major sites of multiplication of the PR8 and Lee B strains of influenza virus in chick embryos injected by the amniotic route were in the cells lining the amnion and in the epidermal and pharyngeal epithelium. Varying amounts of virus were also present in the epithelium of the allantois and less frequently in the peritoneum. No virus was detectable in any of the other tissues of 25 embryos injected between the 7th and 11th days of incubation and examined 48 hours later. Three out of five of the embryos inoculated with the PR8 strain of influenza virus on the 12th day of incubation, on the other hand, showed in addition extensive involvement of the cells lining the respiratory tract. Specific staining of the tissues was first detectable when the ID₅₀ of the amniotic fluids attained a level of greater than 4.5, which corresponded to the time of the appearance of hemagglutinins. With the inocula used this was generally achieved sometime between the 18th and 24th hour of the infection with the PR8 strain of virus and between the 24th and 48th hour of the infection with the Lee B strain of virus. Cytologically, the multiplication of the influenza viruses was characterized by a diffuse type of immunospecific staining which was first detectable in the nuclei and later in the cytoplasm of the cells. The infection progressed rapidly and despite the restricted distribution of the viruses resulted in the death of the embryo in from 3 to 6 days. The results obtained in the present experiments are compared with the findings previously reported in similar studies of mumps virus (6).     

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.     

Characteristics of infection by RSV. III. Duration of the latent period in unsynchronized chick embryo fibroblasts.

The duration of the latent period of RSV was studied in unsynchronized chicken cells using two methods. First, RAV1-producing cells were infected with Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV;subgroup D), and new virions of SR-RSV (subgroup A) produced after phenotypic mixing with RAV1 were neutralized with an antiserum to RAV1. Secondly, uninfected chicken cells were infected with SR-RSV (D) in the presence of either arabionsylcytosine, actinomycin D or cycloheximide, to inhibit the virus production. In both methods, the early decrease of the virus titer showed that new virions were produced as early as 45 min after the beginning of the infection. Earlier times were not explored.     

Reduction of 51Cr-permeability of tissue culture cells by infection with herpes simplex virus type 1.

Infection of different strains of tissue culture cells with herpes simplex virus type 1 (HSV-1) resulted in a reduced 51Cr-permeability. A stability of the cellular membrane to Triton X-100, toxic sera and HSV-specific complement-mediated immune-cytolysis could be observed simultaneously. The results differed with respect to the cell strain used in the experiments.     

Studies on the psittacosislymphogranuloma group. III. The effect of aureomycin on the propagation of virus in the chick embryo

The findings presented indicate that aureomycin could become associated with tissue of the chick embryo by both hematogenous distribution and direct adsorption. Treatment of chick embryos infected with MP virus with 1 mg. of aureomycin by the allantoic route caused an inhibition of virus growth in the allantoic membrane. The drug had no effect on "inert" virus, and appeared to have little effect on adsorption of virus to host tissue. Complete inhibition of growth during the time interval corresponding to the first cycle of multiplication could be achieved only if the drug was administered within 6 to 8 hours after virus inoculation. Partial inhibition of virus multiplication could be achieved even if the administration was delayed as late as 24 hours after infection. In these experiments the chief role of the antibiotic appeared to be one of virustasis reflected in a prolongation of the latent period (non-infectious phase). The virus was able to resume its growth when a critical low level of the drug in the allantoic membrane was reached. When infectivity titrations were carried out using various tissues and organs of treated and untreated embryos, it was found that no virus was detectable in the brains of treated embryos as late as 192 hours after inoculation of virus. This was in contrast with the findings in allantoic membranes and livers of such embryos; these organs showed virus at 120 and 144 hours, respectively. In untreated controls, virus appeared in membranes at 24 hours, in the liver at 48 hours, and in the brain at 72 hours.     

Hepatitis B virus in tissue culture systems. I. Serial propagation of virus in human macrophages

Human peritoneal macrophages, originating from peritoneal dialysis fluid and growing in vitro, support the growth and serial propagation of hepatitis B virus (HBV). The evidence for virus growth is based on the regular detection of HB core antigen in twenty-one passages of macrophages by a complement fixation test using rabbit anti-HB core serum; detection of HB core and e antigen in the twenty-first passage, using an enzyme-linked immunosorbent assay; the detection of HB antigen in cultured macrophages in the fourteenth passage by an indirect immunofluorescence test using human convalescent serum; and detection of HB core antigen in the twenty-second passage by immunofluorescence test using rabbit anti-HB core serum. No CPE was observed through twenty-one passages. It is believed that this is the first successful cultivation and serial propagation of HBV in a cell culture system in vitro. Practical aspects of this finding are discussed.     

Studies on Japanese encephalitis virus infection of reptiles. I. Experimental infection of snakes and lizards

Experimental infection of four species of snakes, Rhabdophis tigrinus tigrinus, Elaphe quadrivirgata, Elaphe climacophora and Agkistrodon halys, and five species of lizards, Takydromus tachydromoides, Eumeces latiscutatus, Eumeces barbouri, Eumeces marginatus oshimensis and Gekko japonicus, with Japanese encephalitis virus (JEV) was carried out. Evidence of JEV multiplication in snakes was not obtained at least under the conditions used in the present study. All lizards except G. japonicus were infected with JEV by ip injection of virus suspension. The minimum infectious dose for a lizard was around 10(3) MLD50/0.05 ml, and this dose was considered to be proportional to the virus dose which is injected into a host by a vector mosquito at a single bite. Temperature dependence of JE virus growth in the lizards was demonstrated. JEV multiplied slower at 20 degrees C than at 26 degrees C, though the peak titers of viremia were equivalent in both groups of lizards kept at 20 degrees C and 26 degrees C. E. latiscutatus developed viremia with ip injection of a partially attenuated strain, Nakayama NIH which could not infect adult mice by peripheral inoculation. T. tachydromoides and E. latiscutatus were also infected by oral feeding of JEV infected mosquitoes. E. latiscutatus was infected by oral feeding of only one infected mosquito.     

Effects of ethanol on chicks in vivo and on chick embryo tibiae in organ culture.

Hypocalcemia previously reported in rats and dogs following oral administration of ethanol may have been caused by a movement of calcium from blood to bone. This present study was undertaken to determine whether ethanol also causes hypocalcemia in chicks and to investigate the direct effects of ethanol on mineral accretion, glucose metabolism and growth of embryonic chick tibiae in an organ culture system. A high dose of ethanol (6 g/kg body wt) produced hypocalcemia, hypermagnesemia and an elevated hematocrit in chicks. Results in vitro were as follows: 1) 5 to 30 mul ethanol/ml medium produced dose-related increases in bone mineral from 58-440%; 2) lactate production was inhibited at all ethanol levels; 3) increased mineral accretion did not occur in ethanol-treated tibiae when iodoacetate was in the medium, but did occur in mechanically disrupted bones exposed to ethanol; and 4) the ethanol response in bone was directly related to the medium phosphate concentration. The results lead to the following conclusions: 1) ethanol has a direct stimulatory effect on bone mineral accretion and an inhibitory effect on bone glucose metabolism in vitro; 2) viable bone cells and an adequate phosphate supply are necessary for the ethanol response, but tissue integrity is not; and 3) the hypocalcemic effect of ethanol in vivo may at least partially result from ethanol-stimulated bone mineral deposition.     

The effect of poliomyelitis virus on human brain cells in tissue culture

Poliomyelitis virus I, Mahoney strain, affected human brain cells grown in tissue cultures usually causing death of the cells in 3 days. The neurons reacted in different ways to the virus, some died with their neurites extended, others contracted one or more of their neurites. Terminal bulbs were frequently formed at the tips of the neurites when they were being drawn into the cell body. The final contraction of the cell body and the change into a mass of granules were often very sudden. Vacuoles often developed in the neuron. There was no recovery. Astrocytes, oligodendroglia, and macrophages were affected by the virus but not as quickly as the neurons. The age of the tissue culture was not a factor when the cells were in good condition. The age of the individual donor of the brain tissue was a factor; the fetal brain cells appeared to be more sensitive to the virus than the adult brain cells. The fetal neurons often reacted ½ hour after inoculation while the adult neurons reacted more slowly, 2 to 24 hours after inoculation. All these changes seemed to be caused by virus infection because they were prevented by specific antiserum or by preheating the virus.