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Yellow fever virus (YFV) causes 30,000 deaths worldwide, despite the availability of a vaccine. There are no approved antiviral therapies for the treatment of YFV disease in humans, and, therefore, these studies were designed to investigate the anti-YFV properties of T-1106, a substituted pyrazine, in a hamster model of YFV disease. Intraperitoneal (i.p.) treatment with 100 mg/kg of body weight/day of T-1106 starting 4 h prior to virus inoculation and continuing twice daily through 7 days post-virus inoculation (dpi) resulted in significantly improved survival, alanine aminotransferase levels in the serum, weight gain, and mean day to death. Virus titer in the liver at 4 dpi was significantly reduced in treated animals, as determined by both quantitative real-time PCR and infectious cell culture assay. No toxicity (weight loss or mortality) was observed at a dose of 100 mg/kg/day in sham-infected control animals. The observed minimal effective dose of T-1106 was 32 mg/kg/day administered either by oral or i.p. treatment. Therapeutic treatment was effective in significantly improving survival when T-1106 was administered beginning as late as 4 days after virus challenge with twice-daily treatment for 8 days at a dose of 100 mg/kg/day. With favorable safety, bioavailability, and postviral challenge treatment efficacy, T-1106 was effective in the treatment of disease in hamsters infected with YFV and should be further studied for potential use as a therapy for human YFV disease.

Cidofovir (CDV) is an effective drug against viruses of theOrthopoxviridaefamily and is active in vitro against variola virus, the cause of smallpox. However, CDV-resistant poxviruses can be generated by repeated in vitro passage in the presence of suboptimal concentrations of CDV. To determine if mutations in the E9L polymerase gene could confer resistance to this nucleoside analog, this gene was sequenced from CDV-resistant vaccinia virus and found to encode five amino acid changes, centered on an N-terminal region associated with 3′→5′ exonuclease activity. Transfer of this mutant E9L gene into wild-type vaccinia virus by marker rescue sufficed to confer the resistance phenotype. E9L polymerase mutations occurred sequentially during passage in CDV, and an H296Y/S338F double mutant that conferred an intermediate CDV resistance phenotype was identified. In vitro, the marker-rescued CDV-resistant vaccinia virus containing all five mutations grew nearly as well as wild-type vaccinia virus. However, the virulence of this virus for mice was reduced, as 10- to 30-fold more CDV-resistant virus than wild-type virus was required for lethality following intranasal challenge. Cidofovir and hexadecyloxypropyl-cidofovir gave partial protection to mice infected with the virus when used at 50 and 100 mg/kg of body weight given as single treatments 24 h after virus exposure, whereas 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) was completely protective at 25, 50, and 100 mg/kg/day when given daily for 5 days. These findings suggest that drug therapy for poxviruses may be complicated by drug resistance but that treatment of the infection with currently known compounds is possible.

Influenza is a highly infectious disease characterized by recurrent annual epidemics and unpredictable major worldwide pandemics. Rapid spread of the highly pathogenic avian H5N1 strain and escalating human infections by the virus have set off the alarm for a global pandemic. To provide an urgently needed alternative treatment modality for influenza, we have generated a recombinant fusion protein composed of a sialidase catalytic domain derived fromActinomyces viscosusfused with a cell surface-anchoring sequence. The sialidase fusion protein is to be applied topically as an inhalant to remove the influenza viral receptors, sialic acids, from the airway epithelium. We demonstrate that a sialidase fusion construct, DAS181, effectively cleaves sialic acid receptors used by both human and avian influenza viruses. The treatment provides long-lasting effect and is nontoxic to the cells. DAS181 demonstrated potent antiviral and cell protective efficacies against a panel of laboratory strains and clinical isolates of IFV A and IFV B, with virus replication inhibition 50% effective concentrations in the range of 0.04 to 0.9 nM. Mouse and ferret studies confirmed significant in vivo efficacy of the sialidase fusion in both prophylactic and treatment modes.

We examined RWJ-270201 in a lethal model of influenza in BALB/c mice. The aim was to delineate the pharmacodynamically linked variable for the drug. Challenge was performed with influenza virus A/Shongdong/09/93 (H3N2). Treatment was administered by gavage. Five doses (1 to 10 mg/kg of body weight) and three schedules (every 24, 12, and 8 h) were evaluated with 10 mice per group. There were 39 placebo-treated mice. Drug exposure was evaluated for infected mice. Exposures were calculated after population modeling of all the plasma concentration-time data simulataneously using the NPEM3 program. Evaluation of dose and schedule with Kaplan-Meier analysis and Cox proportional hazards modeling demonstrated that schedule offered no explanatory power relative to dose alone. Evaluation of peak concentration, trough concentration, and area under the concentration-time curve (AUC) by the same methods revealed that AUC was the dynamically linked variable. Again, schedule offered no further explanatory power when included in the model with AUC. This indicates that AUC is the linked variable and that the anti-influenza effect of RWJ-270201 is independent of schedule. We conclude that once-daily dosing of RWJ-270201 should be evaluated in clinical trials of influenza therapy.

We have recently described GS 4071, a carbocyclic transition-state analog inhibitor of the influenza virus neuraminidase, which has potent inhibitory activity comparable to that of 4-guanidino-Neu5Ac2en (GG167; zanamivir) when tested against influenza A virus replication and neuraminidase activity in vitro. We now report that GS 4071 is active against several strains of influenza A and B viruses in vitro and that oral GS 4104, an ethyl ester prodrug which is converted to GS 4071 in vivo, is active in the mouse and ferret models of influenza virus infection. Oral administration of 10 mg of GS 4104 per kg of body weight per day caused a 100-fold reduction in lung homogenate viral titers and enhanced survival in mice infected with influenza A or B viruses. In ferrets, a 25-mg/kg dose of GS 4104 given twice daily reduced peak viral titers in nasal washings and eliminated constitutional responses to influenza virus infection including fever, increased nasal signs (sneezing, nasal discharge, mouth breathing), and decreased activity. Consistent with our demonstration that the parent compound is highly specific for influenza virus neuraminidases, no significant drug-related toxicity was observed after the administration of oral dosages of GS 4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats. These results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.

The oxygen free-radical scavenger recombinant human manganese superoxide dismutase (MnSOD) was studied for its effects on influenza virus infections in mice when used alone and in combination with ribavirin. Mice challenged with influenza A/NWS/33 (H1N1) virus were treated parenterally in doses of 25, 50, and 100 mg/kg of body weight per day every 8 h for 5 days beginning at 48 h post-virus exposure. An increase in mean day to death, lessened decline in arterial oxygen saturation, and reduced lung consolidation and lung virus titers occurred in the treated animals. To determine the influence of viral challenge, experiments were run in which mice were infected with a 100 or 75% lethal dose of virus and were treated intravenously once daily for 5 days beginning 96 h after virus exposure. Weak inhibition of the mortality rate was seen in mice receiving the high viral challenge, whereas significant inhibition occurred in the animals infected with the lower viral challenge, indicating that MnSOD effects are virus dose dependent. To determine if treatment with small-particle aerosol would render an antiviral effect, infected mice were treated by this route for 1 h daily for 5 days beginning 72 h after virus exposure. A dose-responsive disease inhibition was seen. An infection induced by influenza B/Hong Kong/5/72 virus in mice was mildly inhibited by intravenous MnSOD treatment as seen by increased mean day to death, lessened arterial oxygen saturation decline, and lowered lung consolidation. MnSOD was well tolerated in all experiments. A combination of MnSOD and ribavirin, each administered with small-particle aerosol, resulted in a generally mild improvement of the disease induced by the influenza A virus compared with use of either material alone.

Severe combined immunodeficient (SCID) mice were found to be highly susceptible to murine cytomegalovirus (MCMV) infection. Treatment of infected mice with ganciclovir (12.5, 25, and 50 mg/kg of body weight for 10 days) starting 24 h after virus challenge resulted in delays in death by 2 to 8 days, and no animals survived the infection. (S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) was much more potent, with doses of 1, 3.2, and 10 mg/kg/day (for 10 days) increasing the mean survival time by 15 to 30 days. Twenty-day treatments with HPMPC starting 5 days after virus inoculation increased the mean survival time by 24 to 32 days, with once-weekly (50-mg/kg) treatments being equivalent to daily (10-mg/kg) treatments. Delays in the development of liver, lung, and spleen virus titers in ganciclovir- and HPMPC-treated groups correlated with extensions in the mean survival times relative to the survival times of the placebo controls. The two compounds were approximately equally toxic to uninfected BALB/c mice treated for 10 days, causing 80 to 100% mortality after a dose of 150 mg/kg and 0% mortality after a dose of 75 mg/kg. Thus, the relative therapeutic index of HPMPC was 50-fold greater than that of ganciclovir. Recombinant alpha interferon delta 4 alpha 1/alpha 2 (1 x 10(4) and 5 x 10(4) units per mouse per day) and bropirimine (100 and 300 mg/kg/day) provided no protection from the lethal MCMV infection. The severe combined immunodeficient mouse MCMV infection is an important new model that will permit chemotherapy regimens to be studied over several months.

There is a need for the development of effective antivirals for the treatment of severe viral diseases caused by members of the virus families Bunyaviridae and Arenaviridae . The pyrazine derivative T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has demonstrated remarkable antiviral activity against influenza virus and, to a lesser degree, against some other RNA viruses (Y. Furuta, K. Takahashi, Y. Fukuda, M. Kuno, T. Kamiyama, K. Kozaki, N. Nomura, H. Egawa, S. Minami, Y. Watanabe, H. Narita, and K. Shiraki, Antimicrob. Agents Chemother., 46: 977-981, 2002). Here, we report that T-705 is highly active against a panel of bunyaviruses (La Crosse, Punta Toro, Rift Valley fever, and sandfly fever viruses) and arenaviruses (Junin, Pichinde, and Tacaribe viruses) by cytopathic effect and virus yield reduction cell-based assays. The 50% effective concentrations for T-705 ranged from 5 to 30 μg/ml and 0.7 to 1.2 μg/ml against the bunyaviruses and arenaviruses examined, respectively. We also demonstrate that orally administered T-705 is efficacious in treating Punta Toro virus in the mouse and hamster infection models, as well as Pichinde virus infection in hamsters. When administered twice daily for 5 to 6 days, beginning 4 h pre- or 24 h post-Punta Toro virus challenge, a 30-mg/kg of body weight/day dose provided complete protection from death and limited viral burden and liver disease. A dose of 50 mg/kg/day was found to be optimal for treating Pichinde infection and limiting viral replication and disease severity. In general, T-705 was found to be more active than ribavirin in cell-based assays and in vivo, as reflected by substantially greater therapeutic indexes. Our results suggest that T-705 may be a viable alternative for the treatment of life-threatening bunyaviral and arenaviral infections.

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) was inhibitory to four strains of avian H5N1 influenza virus in MDCK cells, with the 90% effective concentrations ranging from 1.3 to 7.7 μM, as determined by a virus yield reduction assay. The efficacy was less than that exerted by oseltamivir carboxylate or zanamivir but was greater than that exerted by ribavirin. Experiments with mice lethally infected with influenza A/Duck/MN/1525/81 (H5N1) virus showed that T-705 administered per os once, twice, or four times daily for 5 days beginning 1 h after virus exposure was highly inhibitory to the infection. Dosages from 30 to 300 mg/kg of body weight/day were well tolerated; each prevented death, lessened the decline of arterial oxygen saturation (SaO2), and inhibited lung consolidation and lung virus titers. Dosages from 30 to 300 mg/kg/day administered once or twice daily also significantly prevented the death of the mice. Oseltamivir (20 mg/kg/day), administered per os twice daily for 5 days, was tested in parallel in two experiments; it was only weakly effective against the infection. The four-times-daily T-705 treatments at 300 mg/kg/day could be delayed until 96 h after virus exposure and still significantly inhibit the infection. Single T-705 treatments administered up to 60 h after virus exposure also prevented death and the decline of SaO2. Characterization of the pathogenesis of the duck influenza H5N1 virus used in these studies was undertaken; although the virus was highly pathogenic to mice, it was less neurotropic than has been described for clinical isolates of the H5N1 virus. These data indicate that T-705 may be useful for the treatment of avian influenza virus infections.

Hemorrhagic fever of arenaviral origin is a frequently fatal infectious disease of considerable priority to the biodefense mission. Historically, the treatment of arenaviral infections with alpha interferons has not yielded favorable results. Here we present evidence that interferon alfacon-1, a nonnaturally occurring bioengineered alpha interferon approved for the treatment of chronic hepatitis C, is active against Pichinde and Tacaribe arenaviruses in cell culture. In the hamster model of Pichinde virus (PCV) infection, interferon alfacon-1 treatment significantly protected animals from death, prolonged the survival of those that eventually died, reduced virus titers, and limited liver damage characteristic of PCV-induced disease. Moreover, interferon alfacon-1 also demonstrated therapeutic activity, to a lesser degree, when the initiation of treatment was delayed up to 2 days post-virus challenge. Despite the observed advantages of interferon alfacon-1 therapy, efforts to stimulate the immune system with the known interferon inducer poly(I:C 12 U) (Ampligen) offered only limited protection against lethal PCV challenge. Taken together, these data suggest that the increased potency of the bio-optimized interferon alfacon-1 molecule may be critical to the observed antiviral effects. These data are the first report demonstrating efficacious treatment of acute arenaviral disease with alpha interferon therapy, and further study is warranted.

Picornaviruses (PV) include human rhinovirus (HRV), the primary cause of the common cold, and the enteroviruses (EV), which cause serious diseases such as poliomyelitis, meningoencephalitis, and systemic neonatal disease. Although no compounds for PV infections have been approved in the United States, pirodavir was one of the most promising capsid-binding compounds to show efficacy in human clinical trials for chemoprophylaxis of the common cold. Susceptibility to hydrolysis precluded its use as an oral agent. We have developed orally bioavailable pyridazinyl oxime ethers that are as potent as pirodavir. Compounds BTA39 and BTA188 inhibited a total of 56 HRV laboratory strains and three clinical isolates as determined by neutral red uptake assay. At concentrations of 50 s) for the two compounds ranged from 0.5 nM to 6,701 nM. The compounds also inhibited EV, including coxsackie A and B viruses (IC 50 = 773 to 3,608 nM) and echoviruses (IC 50 = 193 to 5,155 nM). BTA39 only inhibited poliovirus strain WM-1 at 204 nM, and BTA188 only inhibited poliovirus strain Chat at 82 nM. EV 71 was inhibited by BTA39 and BTA188, with IC 50 s of 1 and 82 nM, respectively. Both compounds were relatively nontoxic in actively growing cells (50% cytotoxic doses, ≥4,588 nM). These data suggest that these oxime ethers warrant further investigation as potential agents for treating selected PV infections.

Cidofovir {[(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] [HPMPC]}-resistant forms of camelpox, cowpox, monkeypox, and vaccinia viruses were developed by prolonged passage in Vero 76 cells in the presence of drug. Eight- to 27-fold-higher concentrations of cidofovir were required to inhibit the resistant viruses than were needed to inhibit the wild-type (WT) viruses. Resistant viruses were characterized by determining their cross-resistance to other antiviral compounds, examining their different replication abilities in two cell lines, studying the biochemical basis of their drug resistance, and assessing the degrees of their virulence in mice. These viruses were cross resistant to cyclic HPMPC and, with the exception of vaccinia virus, to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine. Three of the four resistant cowpox and monkeypox viruses exhibited reduced abilities to infect and replicate in 3T3 cells compared to their abilities in Vero 76 cells. Compared to the WT virus polymers the resistant cowpox virus DNA polymerase was 8.5-fold less sensitive to inhibition by cidofovir diphosphate, the active form of the drug. Intracellular phosphorylation of [3H]cidofovir was not stimulated or inhibited by infection with resistant cowpox virus. In intranasally infected BALB/c mice, WT cowpox virus was 80-fold more virulent than the resistant virus. Cidofovir treatment (100 mg/kg of body weight, given one time only as early as 5 min after virus challenge) of a resistant cowpox virus infection could not protect mice from mortality. However, the drug prevented mortality in 80 to 100% of the mice treated with a single 100-mg/kg dose at 1, 2, 3, or 4 days after WT virus challenge. By application of these results to human orthopoxvirus infections, it is anticipated that resistant viruses may be untreatable with cidofovir but their virulence may be attenuated. Studies will need to be conducted with cidofovir-resistant monkeypox virus in monkeys to further support these hypotheses.

A novel series of cyclopentane derivatives have been found to exhibit potent and selective inhibitory effects on influenza virus neuraminidase. These compounds, designated RWJ-270201, BCX-1827, BCX-1898, and BCX-1923, were tested in parallel with zanamivir and oseltamivir carboxylate against a spectrum of influenza A (H1N1, H3N2, and H5N1) and influenza B viruses in MDCK cells. Inhibition of viral cytopathic effect ascertained visually and by neutral red dye uptake was used, with 50% effective (virus-inhibitory) concentrations (EC 50 ) determined. Against the H1N1 viruses A/Bayern/07/95, A/Beijing/262/95, A/PR/8/34, and A/Texas/36/91, EC 50 s (determined by neutral red assay) of the novel compounds were ≤1.5 μM. Twelve strains of H3N2 and two strains of avian H5N1 viruses were inhibited at

An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α- d - N -acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.

Matrix protein (M1) is a major structural protein of influenza virus, and it inhibits its own polymerase. A 19-amino-acid peptide, corresponding to a zinc finger region of the M1 sequence of influenza virus strain A/PR/8/34 (H1N1), centered around amino acids 148 to 166, was synthesized. This peptide, designated peptide 6, represents a zinc finger which includes a 7-amino-acid loop or finger and a 4-amino-acid tail at the carboxyl terminus, in addition to the 8 amino acids involved in the coordination of Zn. Three experiments were run to evaluate the activity of peptide 6 on infections induced in mice by influenza A/PR/8/34 and A/Victoria/3/75 (H3N2) viruses. Intranasal (i.n.) treatment of the H1N1 virus infection with 30 or 60 mg/kg of body weight/day, three times daily for 5 days, beginning 4 h pre-or 8 h post-virus exposure, was effective in preventing death, reducing the arterial oxygen decline, and inhibiting lung consolidation. Virus titers in the lungs determined on day 5 were reduced by up to 1.5 log10 in treated groups, but considerable variation in the titers of the recovered virus was seen. The H3N2 virus infection was treated i.n. with 30, 60, or 120 mg of peptide 6/kg/day by using the above-mentioned delayed initiation treatment schedule, and similar protection was seen, although lung virus titers were not reduced in the day-5 assay. Peptide 6 was well tolerated at doses up to 60 mg/kg/day. This zinc finger peptide may provide a new class of antivirals effective against influenza virus.

Immunotoxins were constructed by linking immunoglobulins specific for murine cytomegalovirus (MCMV) to deglycosylated ricin A chain. Toxicities toward MCMV-infected and uninfected cells were determined by measuring the inhibition of protein synthesis following a 48-h exposure to immunotoxins commencing 24 h after infection. The 50% inhibitory concentrations ranged from 0.4 to 4 micrograms/ml for infected cells and from 22 to 120 micrograms/ml for uninfected cells. Selectivity indices ranged from 30 to 157. Control immunotoxins, which were constructed identically except that the immunoglobulin moiety had no specificity toward MCMV antigens, had 50% inhibitory concentrations of 50 and 100 micrograms/ml toward infected and uninfected cells, respectively.

Pulmonary disease in mice induced by influenza virus was monitored by measurement of oxygen saturation (SaO2) in blood with a pulse oximeter. The SaO2 declined in inverse proportion to the viral inoculum. The known antiviral agent ribavirin inhibited the SaO2 decline, prevented death, lowered lung consolidation, and reduced the level of recoverable virus. Pulse oximetry is an effective means of monitoring murine influenzal disease and can be used in the study of potential antiviral drugs.

A series of twelve recently synthesized 6-substituted derivatives of 9-β- d -ribofuranosylpurine 3′,5′-cyclic phosphate (RPcMP) were evaluated for in vitro antiviral activity, using inhibition of viral cytopathogenic effect as the primary parameter for evaluation. Inhibition of the development of intra- and extracellular virus titer was used as a secondary criterion with certain viruses. Five derivatives were considered to have significant antiviral activity. 6-Hydroxylamino-RPcMP was active against type 1 herpes simplex, cytomegalo-, and vaccinia viruses. 6-Thio-RPcMP was inhibitory to types 1 and 2 herpes simplex, cytomegalo-, vaccinia, and type 3 parainfluenza viruses. The 6-methylthio derivative was active against types 1 and 2 herpes simplex, cytomegalo-, and vaccinia viruses, and types 1A, 2, 8, and 13 rhinoviruses; alteration of this 6-substitution to 6-ethylthio or to 6-benzylthio weakened the herpes- and vaccinia virus activity of the compound, but each continued to have significant antirhinovirus activity. The effect of time of addition of 6-methylthio-RPcMP to type 1 herpes simplex virus-infected cells was determined; the compound was most active when added prior to the virus. Early removal of the compound from the infected cells markedly reduced its antiviral effectiveness.

Equine abortion virus (EAV)-induced hepatitis in hamsters presents an interesting animal model for the evaluation of drugs possessing anti-deoxyribonucleic acid virus activity. These experiments demonstrate that 9-β- d -arabinofuranosylhypoxanthine 5′-monophosphate (ara-HxMP), a new synthetic, water-soluble, antiviral agent, effectively controls this disease in hamsters with a therapeutic index of ∼60. Ara-HxMP prevented hepatitis-associated deaths in hamsters, reduced the titer of EAV developing in hamsters, and inhibited the increase of serum glutamic pyruvic transaminase in EAV-infected hamsters.

Topical application of 9-β- d -arabinofuranosylhypoxanthine 5′-monophosphate (ara-HxMP) significantly inhibited the development of keratitis induced by types 1 and 2 herpes simplex virus and vaccinia virus in the eyes of rabbits. Parameters for evaluation of efficacy were infectivity (corneal opacity, lesion size, and type), Draize (erythema, conjunctival swelling, and discharge), and reduction in titer of recoverable virus from the eye. When the relative efficacy of the related compounds 9-β- d -arabinofuranosyladenine (ara-A), ara-A 5′-monophosphate (ara-AMP), and ara-Hx was determined against type 1 herpes simplex virus in a parallel experiment, the more water-soluble compounds (ara-HxMP, ara-AMP) were the most effective. The relative efficacy of ara-A was also determined against type 2 herpes and vaccinia virus-induced keratitis. Mortality in rabbits due to central nervous system involvement caused by types 1 and 2 herpes simplex virus was inhibited. Ara-HxMP was not discernibly toxic to the eye at concentrations of at least 20%; efficacy was still discernible with a 0.1% solution.

9-β- d -Arabinofuranosylhypoxanthine 5′-monophosphate (ara-HxMP) significantly controlled the development of encephalitis produced by deoxyribonucleic acid viruses in mice. In most experiments the activities of ara-HxMP and 9-β- d -arabinofuranosyladenine (ara-A) were determined simultaneously. In the intracerebral (target organ) and intravenous therapy experiments, ara-HxMP had a pronounced advantage over ara-A since the water solubility of ara-HxMP enabled it to be used in much higher concentrations. In experiments where the two drugs were administered intraperitoneally or orally they exhibited similar activity. In several intraperitoneal therapy experiments ara-HxMP was tested alone, using various treatment schedules and dosages. In these experiments, efficacy was observed in groups that had treatments initiated as late as 72 h after virus inoculation.

The effects of four ribonucleic acid virus inhibitors were evaluated in cell cultures and in mice to determine inhibitory effects against bluetongue virus and Colorado tick fever virus (CTFV). Test compounds included 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 3-deazaguanine, 3-deazauridine, and 9-(S)-(2,3-dihydroxypropyl)adenine. Ribavirin-2',3',5'-triacetate (ribavirin triacetate) was evaluated in vivo against CTFV. Inhibition of cytopathic effect and plaque reduction were used to evaluate antiviral activity. In cytopathic effect inhibition studies, bluetongue virus was markedly inhibited by 3-deazaguanine and 3-deazauridine in Vero cells with moderate inhibition by the other agents. Ribavirin and 3-deazaguanine markedly inhibited CTFV in MA-104 cells, 3-deazauridine was slightly less active, and 9-(S)-(2,3-dihydroxypropyl)adenine was negative. Ribavirin was less effective in Vero cells against CTFV. When mice were inoculated intracerebrally with CTFV and treated by a single intracerebral injection with drug, ribavirin triacetate increased the number of survivors, 3-deazaguanine increased mean survival time, and ribavirin was negative. Intraperitoneal treatment of infected mice with ribavirin triacetate for 1 week significantly increased the number of survivors and mean survival time, providing strong evidence that the agent is active across the blood-brain barrier.

A sensitive in vivo system was studied for use in evaluating relatively small quantities of antiviral compounds. Swiss mice were injected intracerebrally with 10 LD 50 of type 1 herpes simplex virus (HSV) or vaccinia virus. Test compounds were injected intracerebrally into mice at a standard time interval after virus inoculation. Significant increases in the number of survivors in drug-treated, infected animals as compared to saline-treated virus controls was the criterion for evaluation of antiviral activity. 9-β- d -Arabinofuranosyladenine (Ara-A) was used to determine an optimal time of therapy for this system. Significant survival increases occurred when the drug was injected 3, 6, 12, and 48 hr after HSV inoculation, with maximal effect occurring after the 6-hr treatment interval. Determination of brain virus titer of 6-hr Ara-A- and saline-treated mice at various intervals indicated that treatment with the drug delayed development of detectable levels of HSV by 1 day. 1-β- d -Arabinofuranosylcytosine (Ara-C), trifluorothymidine (TFT), and 5-iodo-2′-deoxyuridine (IDU) were also evaluated with the use of the 6-hr treatment time against HSV and vaccinia virus. Ara-A and TFT increased the number of survivors among mice infected with either virus, whereas Ara-C increased the survivors among HSV-infected, but not vaccinia virus-infected, mice. IDU was inactive against both viruses.

Virazole (1-β- d -ribofuranosyl-1,2,4-triazole-3-carboxamide) is a highly soluble new synthetic nucleoside having significant, reproducible activity against a broad spectrum of deoxyribonucleic acid and ribonucleic acid viruses in vitro. The drug inhibited viral cytopathogenic effects in monolayers of cells infected for 3 days with type 3 adeno, types 1 and 2 herpes, myxoma, cytomegalo, vaccinia, infectious bovine rhinotracheitis, types 1A, 2, 8, 13, and 56 rhino, types 1 and 3 parainfluenza, vesicular stomatitis, subacute sclerosing panencephalitis, Semliki Forest, Newcastle disease, and measles viruses. Hemagglutinin production by influenza A 2 , influenza B, and type 1 parainfluenza viruses in chicken embryo cells was reduced by Virazole treatment. Recoverable intra- and extracellular virus titers were reduced by the drug in experiments with type 1 herpes, vaccinia, type 3 parainfluenza, and vesicular stomatitis viruses. Plaque formation by type 1 herpesvirus was also inhibited by exposure of the infected cells to Virazole. Pretreatment of cells with the compound, followed by its removal before addition of type 1 herpesvirus, severely lessened the antiviral activity; the compound was still moderately effective in reducing the viral effects on the cells when added as long as 22 hr after the virus. Parallel experiments, in which the antiviral activity of a number of known active drugs was compared, indicated Virazole to have at least a comparable degree of activity, and it was also active against a wider variety of viruses than any of these known active materials. The CCED 50 of Virazole to chicken embryo cells was approximately 1,000 μg/ml, although concentrations as low as 10 μg/ml caused slight (15%) inhibition in total cellular protein after 72 hr of incubation.

Topical application of 1-β- d -ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole) significantly inhibited the development of herpetic keratitis in the eyes of rabbits, as determined by both infectivity and Draize scoring parameters. Significant inhibition of the infection was demonstrated with 10% concentrations of Virazole; a 1% solution had a moderate effect, whereas doses of 0.1 and 0.01% had little activity in this system. A 5% concentration of Virazole similarly inhibited vaccinia keratitis in rabbits. Encephalitis-induced mortality in hamsters initially infected intraocularly with herpesvirus was significantly prevented or inhibited by topical application of 5, 10, and 20% concentrations of Virazole. Surviving, treated hamsters had no signs of herpes keratitis. The 20% concentration was the approximate LD 50 in hamsters. Virazole administered subcutaneously or intraperitoneally to mice did not appreciably alter the course of herpes virus- or vaccinia virus-induced encephalitis in these animals, although in a herpesvirus experiment direct injection of the drug into the brains 3 hr prior to virus inoculation resulted in a significant survivor increase.

PR-879-317A (2,3,5,6,7,8-hexahydro-2-phenyl-8,8-dimethoxy-imidazo [1,2a]pyridine) has been found to be a T-cell-selective immunomodulating agent. In the current studies, a series of experiments was designed to determine the potential antiviral activity of this compound in mice infected with murine hepatitis virus. In a comparative antiviral experiment, the activity seen was superior to that of levamisole, a known immunorestorative agent. This activity was characterized by an increase in the 21-day survival frequency, a decrease in hepatic discoloration, a decrease in the amount of infectious virus recoverable from the liver, and normalization of serum glutamic oxalacetate and pyruvate transaminase levels. A comparison of treatment routes indicated the relative efficacies as intraperitoneal greater than per os greater than intramuscular greater than or equal to subcutaneous. Alteration of the treatment schedule markedly affected the antiviral effect; prophylactic or therapeutic treatments once or twice daily for 3 days were usually effective. Single treatments begun 4 h before or 24 h after virus inoculation were highly efficacious. Three treatments administered on alternate days, beginning 48 h before virus inoculation, proved moderately effective. Thrice-daily treatments were ineffective, as were treatments with durations of greater than 3 days. The optimal dosage varied according to the treatment route and dosage schedule. When assessed for direct antiviral activity in vitro, PR-879-317A failed to demonstrate any significant activity against murine hepatitis virus. The positive in vivo activity noted might therefore be the result of immune modulation rather than a direct antiviral effect.

1-β- d -Ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole) was found to possess significant antiviral activity against aerosol-induced or intranasally induced influenza A 0 , A 2 , and B virus infections in mice. Significant protection was achieved by both oral and intraperitoneal routes of administration. Depending upon the level of virus infection, antiviral activity was best observed at the daily dose of 75 mg/kg. The efficacy of the compound was evidenced by an increase in survivor number, prolongation of mean survival time, suppression of lung consolidation, or decrease in hemagglutinin titer in the infected lung samples. The therapeutic value of this synthetic triazole nucleoside was evident as noted by a significant increase in survivor number even if the treatment was started as late as 24 h after infection with an aerosol of influenza A 2 virus.

In cultured cells, Virazole failed to stimulate either interferon or a cellular resistance which continued in its absence. Serum from Virazole-treated mice likewise contained no demonstrable interferon.