Replication of Newcastle Disease Virus

Infection of L cells with Newcastle disease virus (NDV) results in abortive replication, with the production of very few plaque-forming units (PFU) of virus per cell (W. C. Wilcox, Virology 9:30, 1959; K. Paucker et al., Virology 17:301, 1962). It is known that interferon is synthesized by L cells infected with NDV, and the possibility exists that this endogenously produced inhibitor may be responsible for the abortive replication (W. Henle et al., J. Exptl. Med. 110:525, 1959; J. E. Rodriguez et al., J. Virol. 1:1, 1967). With certain ribonucleic acid (RNA) viruses, enhanced viral replication accompanies the inhibition by actinomycin D of interferon synthesis (E. Heller, Virology 21:652, 1963; R. R. Wagner, Trans. Assoc. Am. Physicians 76:92, 1963). In the present study, the influence of actinomycin D on the synthesis of interferon and on the replication of NDV in L cells was tested. Descriptions of the viruses, cell cultures, and methods used for production and assay of interferon have been given elsewhere (J. S. Youngner et al., J. Bacteriol. 92:862, 1966). Actinomycin D was made available through the courtesy of H. B. Woodruff of Merck, Sharp and Dohme. The influence of suppression of interferon production on the replication of NDV was tested in the following manner. L-cell cultures were treated with different concentrations of actinomycin D and then infected with NDV. The effects of the antibiotic on cellular RNA synthesis (3H-uridine incorporation), interferon production, and virus replication are summarized in Fig. 1. As little as 0.01 ,ug of actinomycin D per ml produced a 23% reduction in labeled uridine incorporation and a 50% reduction in interferon titer. At 0.5 ug of actinomycin D per ml, 3H-uridine incorporation was only 8% of that of the control, and no interferon was detectable in the fluids removed from such cultures after infection with NDV. In contrast, NDV replication was not significantly different in control cultures and in cultures treated with up to 0.1 Mug of actinomycin D per ml. Higher concentrations of antibiotic produced a reduction in virus yield which could be explained, at least in part, by a drop in viability of the cells after 24 hr of exposure to the antibiotic. From the results described in Fig. 1, it was concluded that there was no relationship between interferon production and abortive replication of NDV in L-cell cultures, since inhibition of interferon synthesis by actinomycin D in cells infected with NDV did not enhance the yield of infective virus. The possibility existed that undetected or cell-associated interferon, or inhibitors distinct from interferon, were responsible for the low yield of infective NDV. In an attempt to eliminate this possibility, the ability of a superinfecting virus (Semliki Forest virus-SFV) to replicate was tested in cells in which interferon production was suppressed. This experiment was carried out as follows. Cultures of L cells in 2-oz (56 ml) bottles were divided into four groups. Groups 1 and 3 were treated with actinomycin D (0.5 ug/ml) in the usual manner; i.e., antibiotic was present in all fluids until superinfection with SFV; groups 2 and 4 did not receive the antibiotic. After 1 hr at 37 C, groups 1 and 2 were infected with NDV at an input multiplicity of 5; groups 3 and 4 were not infected with NDV. After 24 hr at 37 C, fluids from each group of cultures were withdrawn and were tested for interferon activity. The cells were challenged with SFV (multiplicity of infection = 8) and incubated for an additional 20 hr at 37 C, after which the fluids from these cultures were harvested and tested for SFV infectivity. The results presented in Table 1 show that, when interferon was produced in response to NDV (group 2), SFV replication was effectively suppressed. In contrast, when interferon production was inhibited by actinomycin D (group 1), SFV replication was almost equal to that obtained in the two control groups (groups 3 and 4) in which cells were not exposed to NDV. The difference in the SFV replication