Anthony R. Scala

Inhibitory effect of glutamine and ammonia on replication of influenza virus in ascites tumor cells.

Abstract Glutamine in the presence of bicarbonate or excess phosphate inhibits replication of influenza cell-associated hemagglutinins in Krebs 2 ascites tumor cells. Ascites cells rapidly liberate ammonia from glutamine. Ammonium ion at a concentration of 0.5–1.3 mg. % caused significant suppression of the growth of influenza and Newcastle virus in Krebs 2 cells. Higher concentrations were required for inhibition in Ehrlich ascites cells and chorioallantoic tissue. There appears to be a relation between the formation of ammonia from glutamine and the observed inhibitory effects of this substance. With delayed addition of glutamine or ammonia (1–4 hours after the virus) there was little or no decrease of hemagglutinin formation.

Inhibition by ammonium ion of the growth of influenza virus in chorioallantoic tissue

Abstract Inhibition by ammonium phosphate of replication of complete influenza virus in chorioallantoic tissue was compared with the action of this inhibitor on production of incomplete virus in ascites tumor cells. In chorioallantoic tissue a concentration of NH 4 + five to ten times higher than in ascites tumor cells was required for inhibition. Infectivity and hemagglutinin titers in the supernatant fluids and cells were reduced in parallel by NH 4 + . At lower virus:cell multiplicities the inhibitory concentration (about 250 μg/ml) was independent of the size of the inoculum. In deembryonated eggs very high multiplicities almost completely abolished the effect of NH 4 + . With multiplicities below 1, addition of NH 4 + 1–4 hours after the virus resulted in production of amounts of hemagglutinin proportional to the size of the inoculum, a result indicating conversion from multiple growth cycles to one-step growth. Virus made latent in tissue cultures by NH 4 + over a period of 24 hours replicated normally when NH 4 + was removed by changing the medium. Bicarbonate at the concentration generally used in tissue cultures augmented the inhibitory effect of NH 4 + .

Formation of noninfectious viral hemagglutinins in ascites tumor cells

Abstract The replication of influenza virus in two strains of ascites tumor cells, Ehrlichs and Krebs 2, in vitro and in the mouse peritoneum was compared with growth of this virus in roller tube suspensions of minced chorioallantoic membrane. Extended incubation of tumor cells with influenza or Newcastle viruses resulted in a progressive cell destruction which was related in degree to the size of the inoculum. Titration of extracts from frozen and thawed tumor cells revealed replication of hemagglutinins which was especially marked in tumors in the mouse peritoneum, where there was little or no increase in infectivity titer. The formation of hemagglutinins in Ehrlich tumor cells was not appreciably diminished by anaerobiosis, but was slightly decreased by hypotonic medium or KCN. In roller tube suspensions of Krebs 2 cells, glutamine and lactate as substrates gave a higher yield of intracellular hemagglutinins than did glutamine and glucose. With the glucose medium, exclusion of oxygen favored the growth of virus in K2 cells.

Further observations on the inhibitory effect of myxoviruses on a transplantable murine leukemia.

Summary Immunization of mice with parainfluenza viruses NDV or Sendai increases the oncolytic effect of these viruses when pre-infected leukemic cells are injected into C3H mice. Variations in oncolytic activity between different strains of influenza and parainfluenza viruses were noted, and also between two leukemic tumors induced by the Gross virus. Statolon given before virus-infected cells prevents oncolysis but has no effect when given later. Antiserum to NDV or Sendai (plus complement) shows a cytolytic effect in vitro against leukemic cells infected with these viruses.

Species source of complement in viral-immune and other cytolytic reactions.

Summary Fresh normal rabbit serum was more effective than guinea pig complement in immune cytolytic reactions with inactivated antiviral serum and ascites tumor cells infected with Sendai virus, and also in cytolysis with a mouse histocompatibility antigen and homologous antibody. When the antiviral rabbit serum had not been heated to 56° both guinea pig complement and rabbit complement augmented viral-immune cytolysis. A primary effect of immune cytolysis on the cell membrane was indicated by the appearance in some cells of permeability to dye before respiration was impaired.

Toxic action of cysteine on ascites tumor cells in vitro

Abstract Cysteine, especially in the absence of other substrates, has a progressive toxic action on Ehrlich ascites tumor cells. Measurements of cell survival in roller tube suspensions by the dye exclusion method and by rate of substratestimulated respiration indicated permanent cell damage at cysteine concentrations as low as 10 to 20 μg/ml. Substrates such as glutamine, glucose, and bovine albumin when added with the cysteine partially protected the cells against poisoning. Lowered oxygen tension and FeSO4 at low concentrations had a similar effect. The toxic action of cysteine and some other SH compounds was related to marked or total inhibition of endogenous respiration after an incubation period of one hour, but cysteine-poisoned cells continued to consume O2 in the presence of glutamine and lactate. Washing of cells after exposure to cysteine reduced their endogenous respiration to a much greater extent than did such treatment of normal cells. The participation of a metal ion in the effect of cysteine is indicated by the protective action of versene (EDTA). Oxidative formation of intracellular disulfide groups and loss of ability to oxidize fats are discussed as possible factors in the inhibitory action of cysteine.

Action of Ammonium Ion on an Early Phase of Viral Infection.

Summary Following adsorption of influenza virus by Krebs 2 ascites cells there is a brief period of sensitivity to inhibition of viral replication by ammonia. After an hour at 35 °C addition of ammonium phosphate causes no inhibition. The period of ammonium sensitivity is prolonged by low temperature, cyanide, or 2-4 dinitrophenol, but the presence of glucose in the medium prevents this effect of CN and DNP. The results suggest that the ammonium-sensitive phase of infection is an energy-requiring process which occurs soon after adsorption and penetration, and comprises initial integration of the viral inoculum with cell components.

Viral Growth and Oncolysis in Krebs 2 Cells as Affected by Substrate.

Summary With 3 strains of influenza virus formation of non-infectious cell-associated hemagglutinins in Krebs 2 ascites cells was much greater in a medium containing lactate and glutamine than when glucose was substituted for lactate. Reduction in tumor-producing capacity of cell suspensions by action of influenza virus was correlated with the effect of substrate on HA formation. A marked oncolytic effect was evident in lactate medium at 4 hours, or before the increase in HA. Although Newcastle disease virus was oncolytic at the same concentration as influenza, growth of NDV and its action on tumor-producing capacity were not affected by the substrate.

Further Observations on the Inhibitory Action of Ammonium Chloride on Influenza Virus.

Summary The effect of NH4Cl on viral penetration was measured in chorioallantoic tissue in vitro by neutralizing absorbed virus with antiserum and determining the subsequent 24 hour yield of hemagglutinins. In some experiments conditions for single cycle replication were obtained by leaving the antibody in the supernatant during the entire period of growth and then measuring cell-associated hemagglutinins. Although some reduction of the amount of penetrated virus was demonstrable one hour after adding virus and NH4Cl this later became insignificant. The prevention of viral penetration by NH4Cl was considered too inefficient and irregular to account for the over-all inhibitory effect of NH4Cl on viral replication. Inhibition by NH4Cl of a type B (Lee) strain was comparable with that for Type A and reversibility of inhibition was demonstrated.

Amino Acid Imbalance and Incomplete Viral Replication.

Four amino acids, lysine, leucine, tryptophane, and phenylalanine, at concentrations of 0.5 to 5.0 mg./ml. inhibit the production of influenza viral hemagglutinins and complement-fixing S antigen in Krebs 2 ascites cells suspended in a medium containing only glucose and glutamate as substrates. In Krebs 2 cells no new infectious virus is formed but the hemagglutinins and CF antigen are produced in high yield. The latter antigen is less sensitive than the hemagglutinin to amino acid imbalance. At minimal inhibitory concentrations of these amino acids formation of viral hemagglutinins was reduced to a much greater extent than cellular protein synthesis. Selective reversal of inhibition was demonstrated, except for leucine, by a survey of the effects of adding each of 18 other amino acids at non-inhibitory concentrations. The effective ratios of reversing amino acid to inhibitor varied from 1∶5 to 1∶100. Certain d-amino acids were much less active than the corresponding l-isomers as inhibitors or reversers. The kinetics of inhibition and reversal suggest that the principal effect of amino acid imbalance may be on the formation of enzymes essential to the initiation of viral replication, but, depending on the amino acid and its concentration, other mechanisms af action are possible.