The antiviral activity of ribosomal polynucleotides against encephalomyocarditis virus infection of mice.

Intraperitoneal administration of ribosomal RNA (rRNA) was found to protect mice against subsequent lethal infection by encephalomyocarditis (EMC) virus without induction of detectable amounts of circulating interferon. The nature of this effect was examined in terms of the types of natural polyribonucleotides which could afford such protection. rRNA prepared from E. coli was slightly more effective than chicken liver rRNA which was, in turn, more effective than yeast rRNA. 5S ribosomal RNA was not effective, whereas the slightly smaller 4S transfer RNA was as good as E. coli rRNA, suggesting that molecular size is not the sole criterion for the protective effect. The separated 16S and 23S E. coli rRNAs where each as effective as the unfractionated RNA. Anti-viral activity was lost after complete hydrolysis with alkali and nucleoside monophosphates were also inactive. Digestion of rRNA with pancreatic ribonuclease greatly decreased its antiviral activity whereas digestion with T1 ribonuclease had no effect indicating that fairly short oligonucleotides, but not of random nucleotide sequence, are active components in the protection of mice against infection by EMC virus. In vitro, no antiviral effect against EMC virus infection was observed in treatment of L cells under various conditions.

Protection of mice against encephalomyocarditis virus infection by chemically modified transfer RNAs.

Periodate or nitrous acid treatment greatly decreases the ability of unfractionated Escherichia coli transfer RNA (tRNA) to be aminoacylated by tRNA-synthetases but these treatments do not affect their antiviral activity against encephalomyocarditis virus infection of mice. Bisulphite treatment of E. coli tRNA reduces its ability to be aminoacylated by 20% and has no effect on antiviral activity. Bromine water treatment of tRNA under conditions causing extensive base modifications eliminates aminoacylation and the antiviral activity of E. coli tRNA. Periodate treatment of yeast tRNA does not affect its antiviral activity and nitrous acid treatment increases its antiviral activity to that of E. coli tRNA. The ability to be aminoacylated does not therefore appear to be essential for antiviral activity of tRNA but extensive modification (bromine water treatment) does destroy antiviral activity.

Protection of mice against encephalomyocarditis virus infection by preparations of transfer RNA.

Preparations of bacterial transfer RNA (tRNA), give dose-dependent protection of mice against encephalomyocarditis (EMC) virus infection at up to I mg tRNA per mouse with maximum response when the tRNA is administered around 6 h before infection. Protection occurs with intraperitoneally and intravenously administered tRNA against infections by both these routes. In some experiments significant protection occurs by single treatments of tRNA up to 24 h after infection with virus doses of I X LD100. Some tRNA preparations of eukaryotic origin do not give significant protection. Protection is not a feature of all species of bacterial tRNA; partially purified valine, tyrosine and phenylalanine tRNAs from Escherichia coli are not protective. tRNA treatment does not induce circulating interferon nor does it 'hypo-reactivate' the protective effect of poly (I).poly (C) treatment of mice. Humoral and cell mediated immune responses do not seem to be involved in tRNA mediated protection since first, cytosine arabinoside treatment does not affect protection by tRNA; second, serum from mice treated with tRNA and an EMC vaccine does not protect other mice against infection, and third, mice that survive normally lethal infections as a result of tRNA treatment are generally just as susceptible to re-infection as previously untreated, uninfected mice. Silica treatment abolishes protection of mice by tRNA implying that macrophages are necessary. However, tRNA does not seem to act by clearance of virus particles since vaccination of mice by inactivated EMC virus is not affected by tRNA treatment. These results are considered in relation to the presence of a tRNA-like structure in EMC virus RNA and protection of mice by other single stranded polynucleotides.

Investigation of the anti-viral mechanism of poly I and poly C against encephalomyocarditis virus infection in the absence of interferon induction in mice.

Protection of mice against EMC virus infection by poly C and poly I has already been distinguished from interferon mediated protection in several ways. Transfer of serum from EMC virus infected and poly C or poly I treated mice to donor mice that were then infected shows that the anti-viral effect of the single-stranded polynucleotides is not due to boosting interferon produced by infection itself in the way that inferferon can be 'primed' in vitro. Mice surviving infections of more than I X LD100 as a result of poly C or poly I treatment show no protection against re-infection 15 days after the first infection, indicating no long-term stimulation of immune responses to the virus. Mice treated with an immunosuppressive regime of cytosine arabinoside can be protected against EMC virus infection with poly C and poly I treatment and athymic 'nude' mice can also be protected. The possibility of IgM stimulation by poly C and poly I seems unlikely from experiments in which serum was transferred from mice treated with the polynucleotides and an inactivated EMC 'vaccine' to recipient mice which were then challenged with infectious virus. Protection of mice against EMC virus by the single-stranded polynucleotides is abolished by administration of silica to the mice, implying an involvement of macrophages in the protective effects of poly C and poly I. The possibility that the polynucleotides stimulate clearance of virus particles, at least from immunologically responsive regions of the mouse, has been discounted by the inability of polynucleotide treatment to suppress 'vaccine' mediated protection of mice. These results indicate that macrophages are involved in the anti-viral effects of poly C and poly I either because they inhibit replication of the virus in macrophages or because direct anti-viral properties of macrophages are activated by the polynucleotides.

Anti-viral activity against encephalomyocarditis virus and Semliki Forest virus and acute toxicity of poly I and poly C administered sequentially to mice.

Mice are protected against lethal intraperitoneal and intravenous infection by encephalomyocarditis virus and Semliki Forest virus by sequential treatment with poly I followed by either polyC or poly5-hydroxyC without production of interferon when the treatments are 4 or more hours apart and by the intraperitoneal or intravenous routes. Maximum protection occurs around 4 hours before infection and is still significant 20 hours after infection. Treatments with combinations of other homoribopolynucleotides were not found to be anti-viral. Protection by sequential polyI, polyC treatment of mice is relatively short-lived and does not 'hypo-reactivate' the protective effect of polyI:C and shows approximately half the protective effect of polyI:C. The toxicity of sequential polyI, polyC treatment is lower than that of polyI:C particularly if poly5-hydroxyC is substituted for polyC. Silica treatment of mice indicates that stationary macrophages are required for protection by polyI followed by polyC but an effect on humoral or cell mediated immune responses does not appear to be involved. The effect appears to be a synergism between the protection conferred by polyI or polyC alone.

Anti-viral activity of single-stranded homopolynucleotides against encephalomyocarditis virus and Semliki Forest virus in adult mice without interferon induction.

Single administrations of poly C or poly I are anti-viral against infections of encephalomyocarditis (EMC) and Semliki Forest virus (SFV) in mice but poly U and poly A are not. The degree of protection is dose-dependent and mice which die do so at a later time when untreated controls even in a strain of mouse in which the time of death is not dependent on the dose of virus given. No circulating interferon is found after treating mice with poly C or poly I even at polynucleotide doses which give the same degree of protection as the interferon inducer, poly I:C. Several additional features distinguish the protection by poly C and poly I from interferon induction: the effect is low 24h before infection and maximal 6 h before infection, the effect is short-lived and mice do not show hypo-reactivation to repeated treatment. Limited treatment of mice with poly I:C, interferon or poly C before infection itself results in additional protection when poly C is also administered after infection, indicating that poly C has an effect after onset of virus replication. After infection poly C and poly I are both more effective by the intravenous route but before infection they are most effective when administered by the same route as the virus. Quantitative comparisons of the protective effects of poly C, poly I and the interferon inducer, poly I:C, are possible from dose response curves of the polynucleotides at different times relative to infection and by different routes of administration. The results are considered in relation to the presence of homopolyribonucleotide tracts in the viral genomes and effects on the reticulo-endothelial system of the mice.