- Volume 10, Issue 2, 1971

Semliki Forest virus, inactivated by incubation at 37°, induced considerably more interferon than did the live virus. Infection by live virus produced an earlier termination of interferon synthesis (at 8 hr) which correlated with a profound inhibition of cellular RNA synthesis. Heat-inactivated Semliki Forest virus did not inhibit cellular RNA synthesis until much later and permitted continuation of interferon synthesis for an additional 4 hr, thereby increasing the yield of interferon by about four- to sixfold.

After heating at 37° in minimal Eagle’s medium, the alternating polyriboadenylic-polyribouridylic acid [poly r(A-U)] became much more active in producing cellular resistance to virus infection and interferon production in four different cell lines (human skin fibroblasts, mouse embryo fibroblasts, mouse L 929 cells and rabbit kidney (RK 13) cells). The thermal activating effect was neutralized by pre-treatment of the cells with DEAE-dextran, which increased the antiviral activity of unheated and decreased the activity of heated poly r(A-U) to the same level. Under all conditions tested, the degree of cellular resistance to virus infection closely paralleled the amounts of interferon produced suggesting that interferon production is responsible for the antiviral resistance produced by poly r(A-U).

Primary cell cultures and a line consisting of fibroblast-type cells were obtained from tadpoles of the common frog, Rana temporaria. One cell line was taken through 21 subcultures during 5 months, when it was abandoned due to contamination by an anonymous Mycobacterium which was completely resistant to antibiotics. Cells from the primary cultures supported the growth of three tick-borne arboviruses without any cytopathic effect; Quaranfil, louping ill (two strains), and Langat. One of the virus strains, louping ill 369t2, was serially passaged in the cell cultures 11 times in 11 weeks, during which time there was considerable multiplication of virus.

Cells infected with Newcastle disease virus contained significant amounts of base-paired RNA. Base pairing in this RNA was suggested by its resistance to ribonuclease and by the sharp thermal transition from ribonuclease resistance to sensitivity at 83° in 0.01 m-NaCl. The base-paired RNA sediments heterogeneously with a peak at 35 s.

Preferential labelling of the base-paired RNA after short periods of incubation with [3H]uridine suggests that it may serve as an intermediate in the synthesis of single-stranded virus-specific RNA. The kinetics of [3H]uridine incorporation into the various species of virus-specific RNA are discussed.

When Detroit cells are infected with poliovirus in the presence of 5 µg./ml. of actinomycin, cell-controlled RNA synthesis is blocked and 2-[14C]uridine is incorporated only into poliovirus-specific RNA.

The addition of polyinosinic-polycytidylic acid (5 µg./ml.) under such conditions results in the inhibition of synthesis of 35 s intracellular, infectious poliovirus RNA.

A high molecular weight fraction of poliovirus-specific RNA migrating on polyacrylamide gels, as would be expected of the double-stranded, replicative form of poliovirus RNA, is synthesized to the same extent as in the absence of interferon inducer. This RNA was digested with RNase and DNase and was resistant to the enzymes, confirming that the compound was replicative form poliovirus RNA.

A low molecular weight, virus-induced RNA of 4s and unknown function is also synthesized independently of polyinosinic-polycytidylic acid.

The significance of the differential inhibition in the synthesis of the compounds presumed to be poliovirus RNA and replicative form poliovirus RNA, and the fact that the capacity of polyinosinic-polycytidylic acid to inhibit the synthesis of infectious RNA is insensitive to actinomycin, is discussed.

This paper reports the effects of treatment with double-stranded RNA of Friend disease, a virus-induced murine leukaemia. The salient feature of the disease is a progressive increase in spleen size; death normally results from rupture of the spleen. It was found that the effect of treatment with double-stranded RNA was closely related to the time of treatment relative to infection. Treatment before or in the early stages of infection increased the severity of the disease, but treatment 5 days after infection led to a profound reduction in the severity of the disease, judged by a pronounced reduction or abolition of splenomegaly. Histological examination of this reversal of splenomegaly showed reduction of the Friend cell infiltration and a return to a more normal spleen architecture. Mice in which remissions have been produced have remained free of evidence of splenomegaly for several weeks. The nature of this therapeutic effect is unknown.

Herpes simplex virus, grown in BHK21 cells, was extensively purified by fluorocarbon treatment, ultracentrifugation, sucrose gradient centrifugation and chromatography on calcium phosphate. The purity of the product was assessed by immunodiffusion, electrophoresis in polyacrylamide gels and removal of added radioactive host material from the virus. Purified virus was disrupted with sodium dodecyl sulphate, urea and dithiothreitol, and at least eight polypeptide components were separated by electrophoresis in polyacrylamide gel.

Thirty-five strains of arbovirus were tested for replication in mouse embryo cell cultures; twenty-six of these multiplied and were cytopathogenic. Dengue 1 and dengue 2 may have multiplied without cytopathogenicity. Twenty-three of the viruses were also tested for the production of complement-fixing antigen; twenty-two were positive. Similar end-points were attained using standard inoculum doses in both tubes and mice for parallel titrations of selected viruses. A major advantage of mouse embryo cell cultures is their ready accessibility to all arbovirus laboratories.

In vitro assembly of human adenovirus type 5 was studied using virus precursor materials from extracts of HeLa cells infected in Eagle’s complete and arginine-free medium. Radioisotope studies suggested that factors having assembly activities in vitro were present only in extracts of cells infected in complete medium. Extracts containing these assembly factors were added to extracts of cells infected in arginine-free medium and the mixtures, after incubation in the presence of stabilizing buffer solutions, contained particles of the same density as complete virus particles formed in vivo. The newly assembled products were DNase-resistant and had 1.5 to 2 log. greater infectivity in plaque assays than either of the two extracts alone or in unassembled mixtures. The factor(s) responsible for the assembly activity were found to be associated predominantly with the nuclear fraction of the infected cells and they were produced in greatest amounts in extracts taken at 12 to 16 hr after infection. Assembly activity of the extracts was lost when they were subjected to temperatures above 32°, trypsin treatment and fluorocarbon extraction before adding them to experimental assembly mixtures.