Nowell Stebbing

Anti-viral activity against encephalomyocarditis virus and Semliki Forest virus and acute toxicity of polyI and polyC administered sequentially to mice

SummaryMice are protected against lethal intraperitoneal and intravenous infection by encephalomyocarditis virus and Semliki Forest virus by sequential treatment with polyI 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.

Inhibition of influenza virion transcriptases by polynucleotides

Abstract Poly(A), poly(I), poly(U), and a thiolated derivative poly(4-thiouridylic acid) inhibit the virion-associated transcriptase of influenza A/Victoria/75 with IDS 50 values of 300, 40, 4, and 1 μg/ml, respectively. A copolymer containing 9% cytidine and 91% 4-thiouridine residues (poly(C,S 4 U 10 )) inhibits the transcriptase to the same extent as poly(4-thiouridylic acid) and the copolymer also inhibits the transcriptase from a range of A strain influenzas, B/Lee/40 and fowl plague virus. Inhibition by poly(U) is dependent on the polymeric nature of the compound: inhibition decreases with oligomers of 20 residues and less. A sugar-phosphate backbone appears to be important for inhibition since vinyl analogs of poly(U) and poly(A) have little or no inhibitory activity.

IN VIVO ANTIVIRAL ACTIVITY OF POLYNUCLEOTIDE MIMICS OF STRATEGIC REGIONS IN VIRAL RNA

Antiviral effects of polynucleotides, particularly double-stranded complexes, mediated by interferon induction, have been well known for a long time. However, many studies with polynucleotides do not preclude the possibility of antiviral effects by other mechanisms. More recently it has become apparant that inhibition of bacteriophage and oncornavirus replication can be achieved by single-stranded polynucleotides that interact directly with polymerases required for virus r e p l i c a t i ~ n . ~ ~ We suggest that single-stranded polynucleotides may have similar direct antiviral effects in lytic virus infections without induction of interferon. There is in fact an a priori argument for the existence of polynucleotides that should be antiviral without, necessarily, inducing interferon. Specific nucleotide sequences in viral R N A must interact directly with factors in cells to produce new, viral proteins, which in combination with host factors result in replication of the virus. Therefore single-stranded RNA molecules that mimic the regions of viral RNA that interact directly with essential host or viral factors (strategic sequences) should act as inhibitors of virus replication. We report here our attempts to identify such strategic sequences in viral RNA of lytic viruses and to show that the antiviral effects we have observed are not due to known possible mechanisms and could therefore be by mimicking sequences within the viral RNA.

Anti-viral effects of single-stranded polynucleotides against avirulent Semliki Forest Virus infection of mice and avirulent infection of rats with encephalomyocarditis virus

SummarySingle-stranded polynucleotide preparations, which neither induce detectable interferon nor affect immune responses, suppress development of antiviral antibodies in mice infected with an avirulent strain of SFV. On a weight basis the antiviral activity of a mixture of poly(I) and poly(ho5C)-copolymer is greater than that of tRNA and similar antiviral effects are observed against a related virulent strain of SFV. EMC virus causes an avirulent infection of rats and development of EMC virus antibodies (routinely determined by assaying the protective effect of rat serum against EMC virus infection of mice) is suppressed when the rats are treated with tRNA or the mixture of poly(I) and poly(ho5C)-copolymer. This suppression of antibodies to EMC virus appears to reflect reduction of virus replication. Treatments of 6 mg/rat i.p. or i.v. 6 hours before infection confer essentially the same antiviral effect as 3 times these polynucleotide doses administered during 3 days immediately post infection. These results with avirulent infections indicate that the previously reported antiviral effects of the single-stranded polynucleotides are not simply due to modifications of the tissue pathology which leads to death in the case of virulent virus infections.

Antiviral effects of single-stranded polynucleotide inhibitors of the influenza virion-associated transcriptase against influenza virus infection of hamsters and ferrets

Administration of a single-stranded polynucleotide copolymer containing 9% cytidine residues and 91% 4-thiouridine residues [poly(C,S4U10)], a known potent inhibitor of the virion transcriptase of influenza viruses, suppressed the amount of virus recoverable from the nasal washes of influenza virus-infected hamsters and ferrets. The incidence of sneezing and nasal discharge in infected ferrets was also reduced. In hamsters, poly(C,S4U10) was more effective than amantadine-HCl or Virazole. Polyinosinic acid in combination with poly-5-hydroxy cytidylic acid also had anti-influenza effects. Poly(C,S4U10) annealed to polyadenylic acid was not effective, nor was the double-stranded polymer (polyinosinic acid) . (polycytidylic acid) even when complexed with carboxymethylcellulose and polylysine. No toxic effects of poly(C,S4U10) were apparent in the treated hamsters and ferrets, and high doses (greater than or equal to 2.86 g/kg) administered intraperitoneally to mice produced no adverse effects.

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

SummaryIntraperitoneal 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 fromE. 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 asE. coli rRNA, suggesting that molecular size is not the sole criterion for the protective effect. The separated 16S and 23SE. coli rRNAs where each as effective as the unfractionated RNA. Antiviral 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.

Studies on the antiviral activity of 5′-amino-2′,5′-dideoxy-5-iodouridine (AIU) against herpes viruses in vivo and in vitro

Abstract 5′-Amino-2′,5′-dideoxy-5-iodouridine (AIU) is known to have antiviral activity against herpes simplex virus type 1 (HSV-1) in cell cultures but is less potent than the parent compound 5-iodo-2′-deoxyuridine (IDU). Studies on its activity in vivo are limited. AIU showed antiviral effects in BHK cells against wild-type HSV-1 but not a thymidine kinase-negative (TK − ) mutant, indicating the importance of phosphorylation of the compound by HSV-1-induced thymidine kinase for antiviral effects. When cells were coinfected with the TK − mutant and a wild-type TK + strain, both strains were inhibited by AIU, suggesting that the failure to phosphorylate AIU accounts for the resistance of the TK − strain alone. AIU failed to limit death after systemic HSV-1 infection of mice when the drug was administered parenterally or orally, although IDU in similar treatment regimens was effective. Tested for efficacy in a local HSV-1 skin infection of mice, topical AIU in a petrolatum ointment or dimethyl sulphoxide (DMSO) did not reduce titres of virus in the skin or modify the clinical course of infection, whereas topical application of IDU in DMSO caused a significant reduction in the titres of virus in the skin and reduced both the occurrence and the severity of lesions. When administered subcutaneously or orally AIU had a slight antiviral effect against HSV-1 infection in the skin of mice. Moreover, intraperitoneally (i.p.) administered AIU limited HSV-1 replication in peritoneal cells of i.p. infected mice, indicating that AIU is inherently antiviral in vivo. The poor antiviral activity of AIU in vivo compared with IDU is attributed to its lower antiviral potency as judged by its activity in cell cultures and its inability to enter neural tissue.

Studies on the mode of action of single-stranded polynucleotides which are antiviral against encephalomyocarditis virus infection of mice

SummaryPoly(dI) and poly(dC) administered separately or sequentially show no antiviral effects against EMC virus infection of mice, whereas poly(rI) and poly(rC) are antiviral in such treatment regimens without evidence of interferon induction. The antiviral effects of poly(rI) and poly(rC) appear to depend on single-strandedness because their antiviral effects are decreased by annealing to poly(dC) and poly(dI) respectively. This decrease in antiviral effect would not seem to be due to an adverse effect of polydeoxyribonucleotides on EMC virus infection because the polydeoxyribonucleotides have no effect on the antiviral activity of another single-stranded RNA,E. coli tRNA.

The Direct Anti-Viral Activity of Single Stranded Polyribonucleotides. II. The Effect of Molecular Size and the Involvement of Cellular Receptors

We have examined the dependence on molecular size of various singlestranded polynucleotides that protect mice against encephalomyocarditis (e. m. c.) virus infection in the absence of circulating interferon. Sequential treatment of mice with various sizes of poly(I) at 8 h before infection followed by poly(C) at 4 h before infection conferred highly significant protection against e. m. c. virus infection. At 100 μg/mouse of both the polynucleotides significant protection occurred with all the sizes of poly(I) examined but the degree of protection decreased with poly(I) smaller than S 20, w of 6.13. In this case a decrease in the size of the poly(C) component lowered the degree of protection. On lowering the polynucleotide doses to 20 μg/mouse, significant protection was achieved only when the poly(I) had an S20, w value equal to or greater than 4.39 and again the degree of protection conferred was related to the size of poly( C). Mixtures of poly(I) and a copolymer containing 5-hydroxycytidylic acid, here designated poly(ho5C) copolymer, also conferred significant protection against infection at 8 h before infection providing that the two components were sufficiently large. We only examined the anti-viral activity of mixtures containing equal masses of poly (I) and the poly(ho5C) copolymer at a dose of 20 μg/mouse of each polynucleotide. With poly(l) of all sizes examined (S20,W between 2.50 and 12.5) significant protection occurred when the poly(ho5C) copolymer had an S20,W value of 8.29 or greater. With poly(ho5C) copolymer preparations of progressively smaller sizes significant protection only occurred with progressively larger sizes of poly(I). These results are considered in relation to other phenomena, including interferon induction by the double stranded complex, poly(I:C), showing dependence on the size of poly(i).

The direct anti-viral activity of single stranded polyribonucleotides I. Potentiation of activity by mixtures of polymers which do not anneal

Mixtures containing equal masses of polyinosinic acid (poly (I)) and copolymers containing poly(5-hydroxycytidylic acid), (poly(ho5C) copolymers), protect mice against encephalomyocarditis (e. m. c.) virus infection and the protective effect is greatest 6 h before infection. The batches of poly(ho5C) copolymer used in these studies were obtained by bromine water treat ment of polycytidylic acid (poly(C)) and contained cytidine, bromocytidine and hydroxycytidine residues with the latter component comprising between 50% and 81% of the total residues in different preparations. These copolymers were used in solution or in a gelled form obtained by freezing in the absence of salt. Evidence is presented which shows that double stranded complexes do not form when the poly(ho5C) copolymers are mixed with poly(I). Protection of mice with mixtures of poly(I) and poly(ho5C) copolymer was slightly greater than had previously been observed with sequential administration of poly(I) followed by poly(C) or the homopolymer form of poly(ho5C). The gelled form of poly(ho5C) copolymer conferred slightly greater protection than poly(ho5C) copolymer used in the soluble state. The percentage of hydroxycytidine residues in the copolymers did not affect the degree of protection conferred by mixtures of the copolymers with poly (I). Administration of mixtures of poly(I) and soluble poly (ho5C) copolymer by the intraperitoneal route was more effective than the intravenous route when high doses were used at 6 h before infection. At this time, 200 μg/mouse of a mixture of poly (I) and poly(ho5C) copolymer was as protective as 60μg/mouse poly(I:C) but the toxicity of mixtures of poly(I) and poly(ho5C) was less than one tenth that of poly(I:C). The mechanism of the protective effect with single stranded polynucleo tides cannot yet be stated with confidence. However, the protection by mixtures of poly(I) and poly(ho6C) copolymer does not seem to be through interferon production. Serum from mice treated with the nurture of polynucleotides cannot confer protection on other mice subsequently exposed to infection. Further, the mixtures do not hypo-reactivate the protective effect of poly (I : C) treatment which would have been expected had both effects had a common basis in the interferon phenomenon. Moreover, treatment with the mixture does not result in a stimulation of immune response produced by e. m. c. virus infection.