Interferon production in mice by vesicular stomatitis virus

In considering the characteristics of viruses that stimulate the production of interferon, one generalization that has been made is that avirulent viruses are more effective inducers of interferon synthesis than are virulent viruses (5). In particular, it was found (6) that a highly virulent strain of vesicular stomatitis virus (VSV), which did not itself elicit interferon synthesis in Krebs-2 carcinoma cells, could inhibit interferon production in the same cell line that had been infected with Newcastle disease virus, an effective inducer of interferon synthesis. It was also reported that VSV acted much like actinomycin D, shutting off host cell ribonucleic acid synthesis soon after exposure to the virus. In contrast to these findings, we have observed that VSV is capable of stimulating the formation of interferon in mice, and the present report deals with the production of interferon by virulent and avirulent mutants of VSV. Two plaque-size mutants were isolated from routine passages of the Indiana strain of VSV grown in chick embryo (CE) cell cultures. After five successive clonings, one mutant (LI) formed plaques approximately 4 mm in diameter at 48 hr postinfection and the other mutant (S2) formed plaques approximately 1 mm in diameter. To prepare virus pools, the mutants were grown in CE cell cultures at low multiplicities of infection to avoid the formation of incomplete, interfering components found in VSV preparations grown at high multiplicities of infection (1-3). The lethality of the two plaque-size mutants was measured in Swiss mice (Taconic Farms, females, 25 to 30 g) by injecting groups of animals intracerebrally with 0.03 ml of serial 10-fold dilutions of virus. All deaths were recorded for a 14-day period. The LI mutant was extremely lethal for mice; 4.0 to 7.5 plaque-forming units (PFU) constituted 1 LD5o dose. In contrast, 4.8 X 103 PFU of the S2 mutant was required for 1 LD50 dose. These results are in excellent agreement with those of Shechmeister and his colleagues (4), who worked with similar mutants. The ability of the S2 and LI mutants to stimulate interferon production in mice was investigated by injecting groups of animals intravenously with 0.1 ml of appropriate dilutions of virus; 12 hr later, the mice were bled by cardiac puncture (heparinized syringes). The pooled plasmas were dialyzed for 18 hr at 4 C against glycine-HCl buffer at pH 2 to destroy infective virus particles and were brought back to pH 7 by dialysis against phosphate-buffered saline. The plasmas were assayed for interferon content in L-cell monolayers by a plaque reduction method, with Li VSV as challenge virus (8). Both the S2 and LI mutants produced high titers of circulating interferon; the amounts of interferon produced were roughly proportional to the number of PFU inoculated (Table 1). In addition to being stable at pH 2, the inhibitor in mouse plasma was inactive in CE cell cultures and was sensitive to trypsin. The molecular weight of the interferon was 33,000 as measured by filtration through a Sephadex G-100 column (J. V. Hallum, personal communication). Tests were carried out to determine whether the interferon detected in the plasma was synthesized de novo upon injection of the virus or whether it was released from a preformed state (7). Mice were injected intraperitoneally with 244 mg (per kg) of cycloheximide or with saline, and 1 hr later they were injected intravenously with the Li virus. Plasmas from these mice (and from mice injected only with cycloheximide or saline) were collected at 8 and 12 hr, dialyzed against glycine buffer at pH 2 as described above, and assayed for interferon content. Table 2 shows that the inhibition of protein synthesis was accompanied by a 90%7, decrease in interferon titer of plasma obtained 12 hr after injection of the virus. These results indicate that the interferon produced in mice injected with VSV is a newly synthesized protein.