The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus.

We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure. Specific hybridization was observed only when HSV-1 gB DNA was used as probe. This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome. Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells. DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321. In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport. This sequence partially overlaps the PRV gB homolog coding sequence. We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase. Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins. Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein. All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins. The primary protein sequence for HSV-1 gB regions known to be involved in the rate of virus entry into the cells and cell-cell fusion, as well as regions known to be associated with monoclonal antibody resistance, were highly homologous with the PRV protein sequence. Furthermore, monospecific antibody made against PRV gII immunoprecipitated HSV-1 gB from infected cells. Taken together, these findings suggest significant conservation of structure and function between the two proteins and may indicate a common evolutionary history.

Pyrimidinones. 1. 2-Amino-5-halo-6-aryl-4(3H)-pyrimidinones. Interferon-inducing antiviral agents.

Interferon induction and antiviral activity was discovered with 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone. An analogue study incorporating a series of 2-amino-5-substituted-6-arylpyrimidinones revealed that the most potent interferon inducers were mono- and difluorophenyl analogues. These same analogues were also potent antiviral agents against Semliki Forest virus and herpes simplex type 1. In addition the monomethoxyphenyl analogues were potent antiviral agents but weak interferon inducers. Relatively modest structural changes led to dramatic changes in bioactivity. There was a relatively poor correlation between levels of circulating interferons induced and systemic antiviral activity.

Didemnins A and B. Effectiveness against cutaneous herpes simplex virus in mice.

The antiviral activity of didemnin A and didemnin B against a lethal Semliki Forest virus (SFV) infection of mice and a cutaneous herpes type 1 infection in hairless mice was evaluated. Both compounds significantly decreased the severity of herpesvirus lesions if topical treatment with either didemnin A or didemnin B was started 2 days prior to infection. The survival rate was significantly greater (P = 0.03) in the didemnin B treated group than in controls. If initiation of treatment was delayed until 1 h after infection, no activity was obtained. The compounds were not active against cutaneous herpesvirus infection when injected intraperitoneally (i.p.). Didemnin B at concentrations as low as 1.5 micrograms, administered topically 3 times daily for 5 days, produced skin irritation. Eight times this level of didemnin A could be administered before similar toxicity was observed. The limited activity of didemnins A and B coupled with irritation at the treatment site limits their usefulness in treating cutaneous herpesvirus infection. Neither didemnin A nor B had significant activity in SFV-infected mice.

Comparison between circulating interferon and drug levels following administration of 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) to different animal species.

Using a high-performance liquid chromatographic assay, these studies attempted to correlate circulating levels of 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) with the serum interferon response induced in mice, cats, dogs, cattle, and rabbits. The order of greatest sensitivity for interferon induction by ABPP was mice greater than cats greater than dogs greater than cattle greater than rabbits. Experiments to date indicate that the circulating drug levels associated with a detectable interferon response were 10-15 microgram/ml (mice), 15-30 micrograms/ml (cats and dogs), and 30-50 micrograms/ml (cattle). Whereas rabbits produced large amounts (greater than 10(4) units/ml) of interferon when induced with Newcastle disease virus, we could not demonstrate unequivocally that rabbits were induced by ABPP even when circulating drug levels reached 50 micrograms/ml, or greater. We also observed differences in the pharmacokinetics of ABPP in the different species which may contribute to the differences described for the interferon responses. The data point out the need for cautious selection of animal models for preclinical efficacy evaluation and cautious extrapolation of data from preclinical studies to eventual clinical evaluation.

Interferon induction by and toxicity of polyriboinosinic acid [poly(rI)].polyribocytidylic acid [poly (rC)], mismatched analog poly (rI).poly[r(C12Uracil)n], and poly(rI).poly(rC) L-lysine complexed with carboxymethylcellulose.

The ability of polyriboinosionic acid [poly(rI)].polyribocytidylic acid [poly(rC)], mismatched analog poly (rI).poly[r(C12Uracil)n], and poly(rI).poly(rC) complexed with poly L-lysine and carboxymethylcellulose [poly(ICLc)] to induce interferon and the comparative toxicity of each in cats were evaluated. Each induced high levels of circulating interferon, although poly(ICLC) injected intravenously at 1 to 4 mg/kg induced up to 10 times more interferon than the other compounds. Each compound was pyrogenic and caused a transient decrease in leukocyte numbers. Poly(rI).poly(rC) and the mismatched analog caused severe diarrhea and nausea at the highest drug concentrations (1 to 4 mg/kg), but poly (ICLC) did not. Each compound also caused depression and lethargy and impaired coordination.

Interferon induction by 5-halo-6-phenyl pyrimidinones.

The interferon inducing characteristics of a new series of 6-phenyl pyrimidinol compounds are described and compared against a previously identified pyrimidine, 2-amino-5-bromo-6-methyl-4-pyrimidinol (ABMP). Interestingly, a split in ability to induce interferon but not in vivo antiviral activity was observed in the newest compounds. One representative compound, 2-amino-5-bromo-6-phenyl-4-pyrimidinol (ABPP) induced high levels of serum interferon in mice, cats and cattle in vivo and human lymphoid tissue in vitro and was consistently more active than ABMP. Another representative compound, 2-amino-5-iodo-6-phenyl-4-pyrimidinol (AIPP) was a poor interferon inducer in every system evaluated yet was as active as an in vivo antiviral agent as ABPP or ABMP. The serum interferon response induced by both ABMP and ABPP appeared to originate from an antilymphocyte serum resistant but radiosensitive cell population in the thymus and spleen. These results suggest that the antiviral activity of this group of agents is mediated by both interferon and interferon independent mechanisms.

Antiviral and interferon-inducing properties of 1,5-diamino anthraquinones.

A series of anthraquinones with amino substituents at the 1,5 positions were found to induce interferon in mice. A prototype compound, 1,5-bis[(3-morpholinopropyl)amino]-anthraquinone (Ia), was an effective antiviral agent when administered either orally or parenterally. Peak interferon titers were found 12 to 24 h after drug treatment. The minimum oral dose of Ia required to induce serum interferon or to protect mice against a lethal virus infection was 62 mg/kg. Mice tolerated an oral dose of at least 30 times this minimum effective dose. A single dose of Ia given up to 6 days prior to infection had significant protective activity. Biological properties of Ia were compared with those of three other 1,5-diamino anthraquinones, which also induced interferon and demonstrated antiviral activity in mice. The most active compound was 1,5-bis[[2-(diethylamino)ethyl]amino]-anthraquinone (Ib), which protected mice against virus infection at a dose as low as 8 mg/kg (less than 1/60 its maximum tolerated dose). Mice developed hyporeactivity to interferon induction if the same inducer was injected daily, although by alternating between different inducers the loss of interferon responsiveness could be avoided.

Feline interferon response to 2-amino-5-bromo-6-methyl-4-pyrimidinol (U-25,166).

U-25,166 induced high serum interferon levels in cats at concentrations at least 40 times less than the maximum tolerated dose. Although certain cats responded to U-25,166 by consistently producing higher interferon levels than did other cats, this relationship was not observed if the same animals were injected with Newcastle disease virus or polyriboinosinic:polyribocytidylic acid (poly I:C). Using a biweekly treatment regimen, cats remained responsive to interferon induction by U-25,166 over an 18-week period in which 9 doses of the compound were given. Cats given the compound daily, however, soon became hyporesponsive to interferon induction. Interferon was induced in cats given U-25,166 orally as a suspension or in capsules, but circulating interferon levels were low in cats given the drug by subcutaneous injection.

Efficacy of 5,6-dihydro-5-azathymidine against cutaneous herpes simplex virus in hairless mice.

5,6-Dihydro-5-azathymidine, administered subcutaneously, was active both prophylactically and therapeutically against cutaneous herpesvirus infection of hairless mice. Activity was comparable to that obtained with adenine arabinoside.

Stimulation of murine interferon by a substituted pyrimidine.

2-Amino-5-bromo-6-methyl-4-pyrimidinol (U-25,166) induced high levels of circulating interferon in mice when administered either parenterally or orally. Peak titers of interferon were found in the serum between 6 and 12 h after inoculation of the drug. Lower but significant levels of interferon were found in rat serum after administration of U-25,166 by either the intraperitoneal or oral route, and good levels of circulating interferon were observed in cats after oral treatment. Repeated intraperitoneal doses (50 mg/kg) of U-25,166 protected mice against intranasal encephalomyocarditis virus challenge. The minimal effective acute oral dose for antiviral activity was approximately 250 mg/kg. This was also the minimal dose that produced detectable levels of interferon. Maximum tolerated doses in mice were four to six times the minimal effective doses. A single oral treatment was protective in mice against challenge virus inoculated 24 h later. The compound protected mice from challenge with high levels of encephalomyocarditis virus, up to 20,000 mean lethal doses. Antiviral activity in mice was retained when certain minor substitutions were made in the U-25,166 structure.

Recurrent cutaneous herpes simplex in hairless mice.

Passively immunized hairless mice were inoculated cutaneously with herpes simplex virus. Thirty-nine days later, when the primary cutaneous lesions had completely healed, the mice were treated subcutaneously with prednisone. Within 12 to 30 days after starting prednisone treatment, herpesvirus was recovered by skin swabs from 12 of 71 (17%) of the treated mice. This new model has potential application for understanding and treating recurrent cutaneous herpes infections.