Factors Affecting the Sensitivity of Different Viruses to Interferon

When the sensitivities to interferon of Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) were compared by the plaque reduction method in chick embryo cell cultures, NDV was found to be 45-fold more resistant than VSV. This difference was exaggerated when a multiple-cycle yield inhibition method was employed. In marked contrast, when the same viruses were tested by a single-cycle yield inhibition method, the difference in sensitivity to interferon of the two viruses was virtually eliminated. Further investigation showed that, in chick embryo cells exposed to interferon, the resistance to NDV decayed more rapidly than resistance to VSV. This finding explained the divergent results obtained with the two viruses when single- or multiple-cycle replication techniques were employed. Experiments carried out with L cells showed that cellular antiviral resistance decayed much more slowly in these cells than in chick embryo cells. Consequently, when measured by the plaque reduction method in L cells, no difference was observed in the sensitivity to interferon of VSV and NDV(pi), a mutant of NDV which replicates efficiently in L cells. A procedure is suggested for determining the relative sensitivities to interferon of different viruses under conditions which minimize the role of decay of antiviral resistance in the host cells.     

Effect of Interferon on Deoxyribonuclease Induction in Chick Fibroblast Cultures Infected with Cowpox Virus

An exonuclease which degrades native deoxyribonucleic acid at pH 9.2 was induced in chick embryo fibroblast cultures and in human amnion cells by infection with cowpox virus. Highly purified chick embryo interferon suppressed the induction of the enzyme in the homologous cell system but not in the human amnion cell cultures. "Mock" interferon prepared from uninfected chicken eggs and purified in the same manner as biologically active interferon preparations had no effect on the induction of the enzyme.     

Antiviral activity of preparations of RNA isolated from cells treated with interferon (messenger RNA for an antiviral protein?)]

Chick embryo cultures treated with interferon yielded a biologically active RNA which, upon inoculation into chick embryo cells, created an antiviral condition in them. The level of vesicular stomatitis virus reproduction in such cells was 2-30% of that observed in the cells treated with control RNA. The maximum activity of the experimental RNA was seen 3 hours after the treatment with interferon.     

Inhibition of Mengo virus by interferon

Gauntt, Charles J. (The University of Texas, Austin), and Royce Z. Lockart, Jr. Inhibition of Mengo virus by interferon. J. Bacteriol. 91:176-182. 1966.-The inhibition of Mengo virus replication in L cells resulting from interferon was studied quantitatively. Interferon was titrated on L cells with Western equine encephalomyelitis (WEE) virus as the challenge virus. One protective unit (PU) of interferon is the least amount of interferon which prevents cytopathic effects when a large multiplicity of WEE virus is added subsequent to overnight incubation with interferon. Ten PU of interferon reduced the yields of Mengo virus by about 90%. Larger doses of interferon, up to 220 PU, caused no further reduction in the amount of virus produced. Plaque formation by Mengo virus was also reduced in number by about 85 to 90%, but could not be further reduced. The plaques which formed on interferon-treated cells were reduced in size. We were unable to obtain a virus population with increased resistance to interferon action by use of five successive growth cycles in interferon-treated cultures. Analysis of the cell population for the proportion of cells able to act as infectious centers revealed that incubation of cells with 10 PU of interferon decreased the proportion of virus-yielding cells by 80%. The yield of virus per virus-producing cell was decreased by 40 to 60%. Despite the reduction in yields, plaques, and infectious centers resulting from interferon, all doses of interferon failed to prevent the complete destruction of the cells. Experiments with puromycin indicated that the cytopathic effects observed in L cells infected with Mengo virus required that a virus-directed protein be synthesized between 4 and 5 hr postinfection. The evidence suggested, therefore, that the Mengo virus genome was able to code for new protein synthesis in the absence of the production of infectious virus.     

Effect of calcium chloride on interferon production and the biosynthesis of cellular macromolecules

The influence of calcium chloride in production of interferon and biosynthesis of cell proteins, RNA and DNA in the cultures of chick embryo fibroblast cells and murine cells L-929 in response to induction of the cells by poly(I).poly(C) was studied. It was shown that calcium ions in concentrations of 10 to 30 mM markedly increased formation of interferon in the cell cultures.     

Effect on drugs changing the intracellular level of adenosine 3',5'-cyclic monophosphate on interferon formation in chick embryo cells of different ages]

The effect of theophylline and adrenaline on the synthesis of interferon induced by the influenza B virus, strain Lee, in a chick embryo tissue culture was studied. Both preparation were found to decrease interferon synthesis when 5-day-old cultures were used; the inhibitory effect was increased when the two drugs were used together. The degree of inhibition of interferon production depended on a dose of the preparation; the inhibition was still present even when the drugs ere introduced several hours after the cells were infected with interferonogen. The treatment of one-day-old cultures with theophylline resulted in increase of interferon synthesis, whereas administration of adrenaline alone or together with theophylline did not affect the level of interferon synthesis. The drugs used produced no effect on the reproduction of the test-virus of vesicular stomatitis, Newcastle disease and Chickungunya viruses in chick embryo cells and influenza B virus in the developing chick embryos. The results obtained are discussed from the point of view of a possible influence of the intracellular adenosine 3',5-cyclic monophosphate level on the synthesis of virus-induced interferon.     

Temperature-sensitive Steps in the Biosynthesis of Venezuelan Equine Encephalitis Virus

In contrast to Eastern equine encephalitis virus, the replication of Venezuelan equine encephalitis (VEE) virus was strongly inhibited at 44 C in chick embryo cells. The inhibited steps were analyzed by shifting the incubating temperatures up or down, and by determining during the shifts the rate and extent of infectious ribonucleic acid (RNA) synthesis, intact virus synthesis, and formation of complement-fixing antigen or of antigen detectable by a direct fluorescent-antibody technique. The inhibition appeared to be due to two temperature-sensitive steps involved in the synthesis of VEE virus in chick embryo cells. The first step of inhibition at 44 C occurred early in virus replication and could be completely reversed simply by transferring cultures to 37 C. The inhibition appeared to take place at some point between the time when the virus entered the cell and was uncoated and the beginning of viral RNA synthesis. The second temperature-sensitive step in VEE virus synthesis was irreversible; it occurred at a point after the synthesis of viral RNA, and before the formation of virus protein measured as complement-fixing antigen or as antigen that could be stained with fluorescent antibody.     

Viral Ribonucleic Acid Synthesis by Newcastle Disease Virus Mutants Isolated from Persistently Infected L Cells: Effect of Interferon

The synthesis of different viral ribonucleic acid (RNA) species was studied in chick embryo (CE) and mouse L-cell cultures infected with the Herts strain of Newcastle disease virus (NDV(o)) and a mutant isolated from persistently infected L cells (NDV(pi)). In CE cell cultures, both viruses synthesized significant amounts of 54, 36, and 18S RNA. However, in L cells, synthesis of 54S virion RNA was markedly reduced. From these results, it seems likely that the low yield of infective virus in L cells is due to a deficient synthesis of 54S RNA in this host. On this basis, however, it is apparent that the "covert" replication of NDV(o) in L cells is due to factors other than viral RNA synthesis. When low concentrations of interferon were used to pretreat CE cells, a differential effect on the synthesis of various RNA species was observed. The 18S RNA of NDV(o) was more sensitive to interferon action than the 36 and the 54S RNA species. In contrast, the 18S RNA of NDV(pi) was less sensitive than the 36S and the 54S RNA. The inhibition of 54S RNA synthesis correlated with the reduction of viral yield and explained the greater sensitivity of NDV(pi) to interferon.     

Viral Events Necessary for the Induction of Interferon in Chick Embryo Cells

Temperature-sensitive mutants of Sindbis virus were employed to investigate the nature of the viral event(s) which induces chick-embryo cells to produce interferon. Chick embryo cells induced by the parental heat-resistant strain of Sindbis virus produced essentially equal amounts of interferon at 29 and 42 C. An RNA⁻ and three RNA⁺ strains [temperature-sensitive mutants unable (RNA⁻) and able (RNA⁺) to make ribonucleic acid] produced interferon at 29 C but not at 42 C. It is concluded that viral RNA per se and the replication of viral RNA do not induce interferon production by chick embryo cells.     

Studies on cytotropism in animal viruses. 3. Growth of influenza virus in epithelial-like and fibroblastic cells derived from chick embryo lung

Stewart, Robert B. (University of Rochester, Rochester, N.Y.), and Paul H. Frickey. Studies on cytotropism in animal viruses. III. Growth of influenza virus in epithelial-like and fibroblastic cells derived from chick embryo lung. J. Bacteriol. 92:972-977. 1966.-Growth of the PR8 strain of influenza virus was studied in epithelial-like and fibroblastic cell cultures derived from chick embryo lungs. The cells were found to differ in morphology, staining characteristics, and in their ability to support production of infectious influenza virus. Fibroblastic cells were characterized by their spindle shape, content of a mucopolysaccharide, their relative inability to synthesize infectious influenza virus, and production of a cell-associated noninfectious hemagglutinin. Epithelial-like cells were characterized by their polygonal shape, absence of mucopolysaccharide, and ability to synthesize infectious influenza virus.     

Comparative Immunogenicities of Chikungunya Vaccines Propagated in Monkey Kidney Monolayers and Chick Embryo Suspension Cultures

A comparative study was made of Formalin-inactivated Chikungunya vaccines prepared from the virus propagated in African green monkey kidney monolayers and concentrated chick embryo suspension cultures. The vaccine prepared in the chick embryo suspension cultures was significantly more protective to mice against a live homologous virus challenge and stimulated the production of 4 to 5 times more circulating antibodies than the vaccine prepared with virus grown in African green monkey kidney monolayer cultures.     

Isolation and identification of Marek's disease virus by fluorescent antibody technique.

Cell-associated herpesvirus related to Marek's disease (MD) was isolated from the direct culture of kidney cells of naturally infected chickens at Taoyuan or by inoculation of clinical specimens to chick kidney (CK) and chick embryo fibroblast cells. The virus isolates replicated in CK or chick embryo kidney cell cultures were identified to be MD by the fluorescent-antibody technique.     

Cells Persistently Infected with Newcastle Disease Virus: II. Ribonucleic Acid and Protein Synthesis in Cells Infected with Mutants Isolated from Persistently Infected L Cells

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.     

Effect of mycoplasma on interferon production and interferon assay in cell cultures

Armstrong, D. (The Children's Hospital of Philadelphia, Philadelphia, Pa.), and K. Paucker. Effect of mycoplasma on interferon production and interferon assay in cell cultures. J. Bacteriol. 92:97-101. 1966.-The influence of mycoplasma on the production and action of interferon was studied in cultures of both L and human embryonic kidney (HEK) cells. Mycoplasma hominis 1, the Negroni agent, and the F12 mycoplasma were used for infection of L cells, and M. hominis 1 and M. pneumoniae for inoculation of HEK cells. All strains were capable of multiplication in the culture systems employed. None produced detectable levels of interferon, and responsiveness of the cells to induction of interferon by virus remained unaltered. Infection with mycoplasma did not impair the sensitivity of the cells to the action of interferon, nor was the replication of vesicular stomatitis virus noticeably diminished.     

Long-term cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain (D) of Rous sarcoma virus.

Attempts have been made to keep in vitro, for extended periods of time, cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D. Roller cultures of transformed chick cells kept in serum-deficient medium can be maintained without subcultivation for up to 6 months. The confluent cultures continuously release viruses and viable tumor cells into the medium. The released cells can be plated and have characteristics of growth and morphology which are relatively stable with time until the culture degenerates. Cells released at later stages of the culture produced substantially more viruses than those released earlier, suggesting that cell selection or differentiation occurs during long-term cultivation in low serum concentration. Long-term cultures of untransformed chick embryo fibroblasts can also be maintained in the same way. The release of viable cells by these confluent cultures, however, is negligible.     

Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero)

Vero cells, a line of African green monkey kidney cells, failed to produce interferon when infected with Newcastle disease, Sendai, Sindbis, and rubella viruses, although the cells were sensitive to interferon. Further, infection of Vero cells with rubella virus did not result in interference with the replication of echovirus 11, Newcastle disease virus, or vesicular stomatitis virus, even in cultures where virtually every cell was infected with rubella virus. Under the same conditions, BSC-1 cells and other cells of primate origin produced interferon and showed rubella virus interference. The results indicate that the presence of rubella virus in the cell does not in itself exclude multiplication of other viruses and that rubella virus interference appears to be linked to the capability of the cell to produce interferon.     

Response of Children to Inactivated Measles Vaccine Prepared in Bovine Renal Cell Culture

Formalin-inactivated, alum-adsorbed measles vaccine was readily prepared from virus grown in calf kidney cell culture infected with the Sugiyama strain of measles virus which had been adapted to this cell culture. The vaccine induced no side reaction of any consequence in vaccinated children, but demonstrated antigenic capacity in children as well as guinea pigs, comparable to that of currently used killed measles vaccines prepared from virus grown in monkey kidney or chick embryo tissue cultures. The host system employed for the preparation of this vaccine has an advantage over monkey kidney or chick embryo tissue cultures which are currently used for manufacture of killed measles vaccine. Bovine kidneys are much easier to obtain and cultivate. Of importance is the fact that calf kidneys are practically free of latent virus, whereas monkey kidneys and chick embryos frequently harbor latent viruses.     

Growth of Rio Bravo Virus in Cell Cultures

The growth of Rio Bravo virus (RBV) in eight cell culture systems was studied. Highest yields of virus were produced in BHK-21 (C13), L, and Vero cell lines, but L cells were resistant to low doses of virus. LLC-MK(2), HeLa, and human embryo skin cells produced moderate amounts of virus, but FL amnion and primary chick embryo fibroblasts supported little virus growth. Virus was rapidly inactivated by exposure to pH values below 7.0. Single-cycle growth in BHK-21, L, and LLC-MK(2) cell monolayers was characterized by a latent period of about 12 hr followed by rapid virus production that peaked at 36 to 48 hr. Vero cell cultures can remain chronically infected with RBV for more than 100 days. Such cultures show evidence of cell destruction, and their supernatant fluids contain virus at 10(4) to 10(5) log(10) per ml.