Antiviral activity and mode of action studies of ribavirin and mycophenolic acid against orthopoxviruses in vitro.

Two inhibitors of cellular inosine monophosphate dehydrogenase, mycophenolic acid (MPA) and ribavirin, were evaluated for inhibitory activity against orthopoxviruses. Unrelated antipoxvirus agents tested for comparison included 6-azauridine, cidofovir (HPMPC) and cyclic HPMPC. MPA inhibited camelpox, cowpox, monkeypox and vaccinia viruses by 50% in plaque reduction assays at 0.2-3 microM in African green monkey kidney (Vero 76) and mouse 3T3 cells. Ribavirin was considerably more active in 3T3 cells (50% inhibition at 2-12 microM) than in Vero 76 cells (inhibitory at 30-250 microM) against these viruses. In cytotoxicity assays, MPA and ribavirin were more toxic to replicating cells than to stationary cell monolayers, with greater toxicity seen in 3T3 than in Vero 76 cells. The superior antiviral potency and increased toxicity of ribavirin in 3T3 cells was related to greater accumulation of mono-, di- and triphosphate forms of the drug compared with Vero 76 cells. For both MPA and ribavirin, virus inhibition was closely correlated to the extent of suppression of intracellular guanosine triphosphate (GTP) pools. Treatment with extracellular guanosine (which restored intracellular GTP levels) did not lead to complete reversal of the anticowpox virus activity of ribavirin. This suggests that other modes of virus inhibition also appear to contribute to the anti-orthopoxvirus activity of ribavirin. Biological differences in mode of action and immunosuppressive potential between ribavirin and MPA may account for why the former compound is active against orthopoxvirus infections in animals and the latter inhibitor is not.

Haematological, biochemical and coagulation changes in mice, guinea-pigs and monkeys infected with a mouse-adapted variant of Ebola Zaire virus.

Ebola Zaire virus from the 1976 outbreak (EBO-Z) was recently adapted to the stage of lethal virulence in BALB/c mice through serial passage. In the present study, various parameters were examined in groups of mice and guinea-pigs and in three rhesus monkeys after infection with mouse-adapted EBO-Z. The virus caused fatal disease not only in mice but also in guinea-pigs, in which the course of illness resembled that produced by guinea-pig-adapted EBO-Z. Mice, guinea-pigs and monkeys showed similar haematological and biochemical disturbances, but coagulopathy was less striking in mice than in the other two species. The virus caused severe illness in all three monkeys, one of which died. In the lethally infected monkey the degree of viraemia and the haematological, serum biochemical and coagulation changes were greater than in the other two animals, an observation that may prove to be of value in predicting fatal outcome. All three monkeys developed disseminated intravascular coagulation. The two survivors were completely resistant to challenge one year later with non-adapted EBO-Z. In general, the clinical and pathological changes produced in the three species resembled those previously described in guinea-pigs and non-human primates infected with non-mouse-adapted EBO-Z. It was noteworthy, however, that mouse-adaptation appeared to have resulted in a degree of attenuation for monkeys.

Intracellular phosphorylation of carbocyclic 3-deazaadenosine, an anti-Ebola virus agent.

Carbocyclic 3-deazaadenosine (C-c3Ado) is a potent inhibitor of Ebola virus in mice by infrequent dosing, even though its half life in plasma is only 23-28 min. This prompted studies to determine whether C-c3Ado undergoes intracellular metabolism to derivatives that may promote in vivo activity. In cells, radiolabelled compound readily underwent metabolism to monophosphate, diphosphate and triphosphate (C-c3ATP) forms, with C-c3ATP being the major metabolite detected. A non-polar metabolite was also detected both inside and outside treated cells. The retention time of C-c3ATP was similar but not identical to ATP on a strong anion exchange high performance liquid chromatography (HPLC) column or on a DEAE-Sephadex open column. C-c3ATP and ATP were susceptible to degradation to their respective nucleosides by bovine alkaline phosphatase. Intracellular formation of C-c3ATP reached a plateau by about 4 h after treatment of monkey (Vero 76) and mouse (Balb/3T3 clone A31) cells with 10 or 100 microM extracellular compound. Phosphorylation was linearly dose responsive at 1, 3 and 10 microM. However, the extent of phosphorylation decreased with increasingly higher concentrations (30, 100 and 300 microM). When compound was removed from the medium, the nucleoside cleared the cells within 1 min, whereas C-c3ATP had a half life of decay of 2-3 h in five cell lines. Phosphorylation of C-c3Ado to C-c3ATP was not inhibited by cotreatment of cells (at a 20:1 ratio) with adenosine, guanosine, inosine, xanthosine, cytidine or uridine. There was no evidence of incorporation of C-c3Ado (10 microM) into macromolecules of cells over 72 h, whereas adenosine was readily incorporated. C-c3ATP may represent a form of C-c3Ado that might contribute to extending its intracellular half life or otherwise exhibit antiviral activity and/or toxicity.

Treatment of lethal Ebola virus infection in mice with a single dose of an S-adenosyl-L-homocysteine hydrolase inhibitor.

Ebola Zaire virus causes lethal hemorrhagic fever in humans, for which there is no effective treatment. A variety of adenosine analogues inhibit the replication of Ebola virus in vitro, probably by blocking the cellular enzyme, S-adenosyl-L-homocysteine hydrolase, thereby indirectly limiting methylation of the 5' cap of viral messenger RNA. We previously observed that adult, immunocompetent mice treated thrice daily for 9 days with 2.2-20 mg/kg of an adenosine analogue, carbocyclic 3-deazaadenosine, were protected against lethal Ebola virus challenge. We now report that a single inoculation of 80 mg/kg or less of the same substance, or of 1 mg/kg or less of another analogue, 3-deazaneplanocin A, provides equal or better protection, without causing acute toxicity. One dose of drug given on the first or second day after virus infection reduced peak viremia more than 1000-fold, compared with mock-treated controls, and resulted in survival of most or all animals. Therapy was less effective when administered on the day of challenge, or on the third day postinfection. Single or multiple doses of the same medications suppressed Ebola replication in severe combined immunodeficient mice, but even daily treatment for 15 consecutive days did not eliminate the infection.

A mouse model of aerosol-transmitted orthopoxviral disease: morphology of experimental aerosol-transmitted orthopoxviral disease in a cowpox virus-BALB/c mouse system.

OBJECTIVES: To determine the morphologic changes and disease progression of aerosolized cowpox virus infection in BALB/c mice and to ascertain the suitability of cowpox virus-infected BALB/c mice as a model of aerosol-transmitted, orthopoxviral respiratory disease. METHODS: BALB/c mice were inoculated with cowpox virus, Brighton strain, by aerosol or intranasal route. Mice were killed at specified times after inoculation, necropsied, and tissues were collected for routine histology, immunohistochemistry, and electron microscopy. RESULTS: Inoculation by both routes resulted in disease and death. Immunolabeled viral antigen and lesions predominated in the tissues associated with the inoculation route, that is, lungs, airways, trachea, and nasal passages and sinuses. Tracheitis was evident in the intranasally infected group only. Lesions were generally necrotizing and hemorrhagic, neutrophilic, and increased in extent and severity in a time-dependent fashion. Viral intracytoplasmic inclusion bodies, immunolabeled viral antigen, or virions were readily seen in epithelial tissues, smooth muscle cells of airways and vessels, fibroblasts, periosteal cells, perineural cells, and macrophages. Although the extension of infection appeared to be primarily direct, lesions suggesting hematogenous dissemination were occasionally noted in bone marrow and skin. Transmission electron microscopy demonstrated features of cell injury or death, virion assembly and maturation, and both A-type and B-type inclusions. CONCLUSIONS: Aerosol inoculation of BALB/c mice with cowpox virus provides a reliable and facilitative model of aerosol-transmitted, orthopoxviral respiratory disease.

Cidofovir protects mice against lethal aerosol or intranasal cowpox virus challenge.

The efficacy of cidofovir for treatment of cowpox virus infection in BALB/c mice was investigated in an effort to evaluate new therapies for virulent orthopoxvirus infections of the respiratory tract in a small animal model. Exposure to 2(-5)x10(6) pfu of cowpox virus by aerosol or intranasally (inl) was lethal in 3- to 7-week-old animals. One inoculation of 100 mg/kg cidofovir on day 0, 2, or 4, with respect to aerosol infection, resulted in 90%-100% survival. Treatment on day 0 reduced peak pulmonary virus titers 10- to 100-fold, reduced the severity of viral pneumonitis, and prevented pulmonary hemorrhage. The same dose on day -6 to 2 protected 80%-100% of inl infected mice, whereas 1 inoculation on day -16 to -8 or day 3 to 6 was partially protective. Cidofovir delayed but did not prevent the death of inl infected mice with severe combined immunodeficiency. Treatment at the time of tail scarification with vaccinia virus did not block vaccination efficacy.

Evaluation of immune globulin and recombinant interferon-alpha2b for treatment of experimental Ebola virus infections.

A passive immunization strategy for treating Ebola virus infections was evaluated using BALB/ c mice, strain 13 guinea pigs, and cynomolgus monkeys. Guinea pigs were completely protected by injection of hyperimmune equine IgG when treatment was initiated early but not after viremia had developed. In contrast, mice were incompletely protected even when treatment was initiated on day 0, the day of virus inoculation. In monkeys treated with one dose of IgG on day 0, onset of illness and viremia was delayed, but all treated animals died. A second dose of IgG on day 5 had no additional beneficial effect. Pretreatment of monkeys delayed onset of viremia and delayed death several additional days. Interferon-alpha2b (2 x 10(7) IU/kg/day) had a similar effect in monkeys, delaying viremia and death by only several days. Effective treatment of Ebola infections may require a combination of drugs that inhibit viral replication in monocyte/macrophage-like cells while reversing the pathologic effects (e.g., coagulopathy) consequent to this replication.

Pathology of experimental Ebola virus infection in African green monkeys. Involvement of fibroblastic reticular cells.

BACKGROUND: Ebola virus has been responsible for explosive lethal outbreaks of hemorrhagic fever in both humans and nonhuman primates. Previous studies showed a predilection of Ebola virus for cells of the mononuclear phagocyte system and endothelial cells. OBJECTIVE: To examine the distribution of lesions and Ebola virus antigen in the tissues of six adult male African green monkeys (Cercopithecus aethiops) that died 6 to 7 days after intraperitoneal inoculation of Ebola-Zaire (Mayinga) virus. METHODS: Tissues were examined histologically, immunohistochemically, and ultrastructurally. RESULTS: A major novel finding of this study was that fibroblastic reticular cells were immunohistochemically and ultrastructurally identified as targets of Ebola virus infection. CONCLUSIONS: The role of Ebola virus-infected fibroblastic reticular cells in the pathogenesis of Ebola hemorrhagic fever warrants further investigation. This is especially important because of recent observations indicating that fibroblastic reticular cells, along with the reticular fibers they produce, maximize the efficiency of the immune response.

Antiviral activities of tragacanthin polysaccharides on Punta Toro virus infections in mice.

Tragacanthin polysaccharides from Astragalus brachycentrus (AV208) and Astragalus echidnaeformis (AV212) plants, which are devoid of in vitro antiviral activity, were evaluated in a mouse model of Punta Toro virus (PTV) infection. The PTV (a phlebovirus member of the Bunyaviridae family of viruses) is a model for studying the treatment of Rift Valley fever and hantavirus infections. Single intraperitoneal treatments with 12.5-200 mg/kg/day doses of AV212 given 24 h before or 4 and 24 h after virus inoculation protected the majority of mice from mortality. Single treatments with AV208 were ineffective when given 24 h before the virus challenge; however, protection was afforded when treatments were administered at 4 and 24 h following virus inoculation. In a follow-up study, AV208 treatments of 1.6-50 mg/kg/day given 24 h subsequent to virus exposure caused reductions in mortality, liver infection scores, liver and spleen virus titers, and serum transaminases. The polysaccharides did not activate lymphocytes or natural killer cells, nor was interferon induced in treated mice. However, mice pretreated with fumed silica (a macrophage poison) and infected with the PTV were not protected by subsequent administration of AV208 or AV212 at 50 mg/kg, providing evidence that activation of peritoneal macrophages by the polysaccharides affords protection to infected animals. These compounds should be considered for the potential treatment of significant human infections induced by bunyaviruses and hantaviruses.

Experimental infection of guinea pigs with Venezuelan hemorrhagic fever virus (Guanarito): a model of human disease.

Venezuelan hemorrhagic fever (VHF), a newly described disease caused by an arenavirus (Guanarito), has resulted in multiple human deaths in Venezuela. To develop an animal model of this disease, strain 13 and Hartley strain guinea pigs were inoculated subcutaneously with Guananto strain 95551 of arenavirus in a pilot study to determine susceptibility of the species to the virus. All animals were killed when moribund 12-14 days following inoculation. Animals were necropsied and tissues were fixed and examined by both light and electron microscopy. Viral antigen was demonstrated in the tissues by immunohistochemistry at both the light and electron microscopic levels. Lesions were characterized by single cell necrosis of epithelium of the gastrointestinal tract, interstitial pneumonia, lymphoid and hematopoietic cell necrosis, and the presence of platelet thrombi in occasional blood vessels associated with hemorrhage. Viral antigen was demonstrated in lymphoid tissues and macrophages, endothelial cells of multiple organs, pulmonary epithelium, epithelium of the gastrointestinal tract, and in miscellaneous other tissues and cells. Intact virions and typical arenavirus inclusions were demonstrated by immunoelectron microscopy in these tissues. Based on these findings, the guinea pig appears to be a valid animal model of the human disease.

Passive immunization of Ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses.

A commercially available immunoglobulin G (IgG) from horses, hyperimmunized to Ebola virus, was evaluated for its ability to protect cynomolgus monkeys against disease following i.m. inoculation with 1 000 PFU Ebola virus (Zaire '95 strain). Six monkeys were treated immediately after infection by i.m. infection of 6.0 ml IgG; these animals developed passive ELISA titers of 1:160 to 1:320 to Ebola, two days afer inoculation. However, the beneficial effects of IgG treatment were limited to a delay in onset of viremia and clinical signs, in comparison with untreated controls. The six IgG recipients had no detectable viremia day 5, in contrast with three virus infected controls whose viremias exceeded 7.0 log10 PFU/ml that day. The controls died on days 6, 6, and 7, while two IgG recipients died day 7 and the remaining 4 died day 8, all with high viremias. These results document that passively acquired antibody can have a beneficial effect in reducing the viral burden in Ebola-infected primates; however, effective treatment of human patients may require antibodies with higher specific activities and more favorable pharmacokinetic properties than the presently available equine IgG.

Pharmacokinetics of the antiviral agent 3-deazaneplanocin A.

The pharmacokinetics of 3-deazaneplanocin A (c3Nep), a competitive inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase and novel antiviral agent, was investigated in female BALB/c mice. Animals were given a single intravenous dose of [3H]-c3Nep (0.1 mg/kg; 10 microCi), and blood and selected tissues were collected at various intervals thereafter for up to 72 h. The plasma concentration versus time data for c3Nep was best approximated by a two-compartment open model with first order elimination. The elimination half-life was 12.8 min, the area-under curve (AUC) was 3.38 micrograms.min.ml-1. The distribution of c3Nep into tissues was not extensive. Following 30, 120 min, and 24 h after dosing, the kidneys and the liver contained the highest amount of drug, but this amount did not exceed 1 microgram/g tissue. At these time periods, the majority of activity in the tissues represented labeled derivatives of c3Nep indicating that this compound was converted to stable metabolites. The presence of labeled conversion products in the blood confirmed that this drug is metabolized in vivo. The fact that c3Nep bound to plasma proteins in vitro may explain this drug's limited tissue distribution. The half-life and tissue distribution of c3Nep were different from those of carbocyclic 3-deazaadenosine, a related adenosine nucleoside antiviral drug and AdoHcy hydrolase inhibitor.

Early analysis of viremia and clinical tests in patients with epidemic hemorrhagic fever.

We analysed the early viremia and clinical tests in 82 patients with epidemic hemorrhagic fever (EHF). The results showed that the changes in viremia and clinical tests are related to the severity of the disease and prognosis. Higher concentrations of the virus in infected patients might cause a more unfavourable prognosis and more abnormalities in clinical tests. CK-MB, SGOT, SGPT, serum creatinine and urea nitrogen contents increased markedly, while serum total protein, albumin and calcium contents decreased markedly, indicating that the heart, liver and kidney in EHF patients were severely damaged. Markedly increased WBC and monocytes showed that the patients were seriously infected. Platelet count, antithrombin-III and plasminogen decreased markedly, demonstrating that there were marked changes in the coagulation-anticoagulation and fibrinolytic system of the EHF patients. Changes in RBC, Hb and HCT contents indicated that the blood in the EHF patients had a higher concentration. This study gives further evidence that EHFV plays an important role in the pathogenesis of EHF.

Pharmacokinetics of the antiviral agent carbocyclic 3-deazaadenosine.

The pharmacokinetics of carbocyclic 3-deazaadenosine (Cc3Ado), a competitive inhibitor of S-adenosyl-L-homocysteine hydrolase and novel antiviral agent, was investigated in female BALB/c mice. Mice were dosed either orally or intravenously with a single dose of 10 mg/kg (10 microCi [3H]Cc3Ado), and blood and tissue samples were taken at selected intervals for 24 hr. The plasma concentration vs. time data for Cc3Ado was best described by a two-compartment open model with first-order elimination. The apparent half-life was 23 and 38 min, for intravenously and orally administered Cc3Ado, respectively. Depending on route, tissue concentrations of Cc3Ado reached their maximum by 120 min, and concentrations of Cc3Ado were greatest in the liver, followed by the kidney, spleen, and stomach. By 24 hr, all tissues contained a similar amount of Cc3Ado. In the plasma, one major labeled metabolite was detected, which increased in concentration over time until about 45 min after dosing. None of the plasma Cc3Ado was protein bound, as assessed by in vitro protein binding analysis. Oral Cc3Ado was about 20% bioavailable. Data from this first investigation of the pharmacokinetics of Cc3Ado indicate that this antiviral agent is rapidly distributed and eliminated from the plasma and that distribution to tissues is widespread and rapid.

Antiviral (RNA) activity of selected Amaryllidaceae isoquinoline constituents and synthesis of related substances.

A series of 23 Amaryllidaceae isoquinoline alkaloids and related synthetic analogues were isolated or synthesized and subsequently evaluated in cell culture against the RNA-containing flaviviruses (Japanese encephalitis, yellow fever, and dengue viruses), bunyaviruses (Punta Toro, sandfly fever, and Rift Valley fever viruses), alphavirus (Venezuelan equine encephalomyelitis virus), lentivirus (human immunodeficiency virus-type 1) and the DNA-containing vaccinia virus. Narciclasine [1], lycoricidine [2], pancratistatin [4], 7-deoxypancratistatin [5], and acetates 6-8, isonarciclasine [13a], cis-dihydronarciclasine [14a], trans-dihydronarciclasine [15a], their 7-deoxy analogues 13b-15b, lycorines 16 and 17, and pretazettine [18] exhibited consistent in vitro activity against all three flaviviruses and against the bunyaviruses, Punta Toro and Rift Valley fever virus. Activity against sandfly fever virus was only observed with 7-deoxy analogues. In most cases, however, selectivity of the active compounds was low, with toxicity in uninfected cells (TC50) occurring at concentrations within 10-fold that of the viral inhibitory concentrations (IC50). No activity was observed against human immunodeficiency virus-type 1, Venezuelan equine encephalomyelitis virus, or vaccinia viruses. Pancratistatin [4] and its 7-deoxy analogue 5 were evaluated in two murine Japanese encephalitis mouse models (differing in viral dose challenge, among other factors). In two experiments (low LD50 viral challenge, variant I), prophylactic administration of 4 at 4 and 6 mg/kg/day (2% EtOH/saline, sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 100% and 90%, respectively. In the same model, prophylactic administration of 5 at 40 mg/kg/day in hydroxypropylcellulose (sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 80%. In a second variant (high LD50 viral challenge), administration of 4 at 6 mg/kg/day (ip, twice daily for 9 days, day -1 to +7) resulted in a 50% survival rate. In all cases, there was no survival in the diluent-treated control mice. Thus, 4 and 5 demonstrated activity in mice infected with Japanese encephalitis virus but only at near toxic concentrations. To our knowledge, however, this represents a rare demonstration of chemotherapeutic efficacy (by a substance other than an interferon inducer) in a Japanese-encephalitis-virus-infected mouse model.

Antibodies to Puumala virus in humans determined by neutralization test.

An assay for detection of neutralizing antibodies to Puumala virus using 96-well microtiter plates (NT-ELISA) was developed and evaluated. The test proved to have similar sensitivity and specificity as an IgG ELISA and indirect immunofluorescence test, when screening 187 sera (with an antibody prevalence rate of 19%) from normal populations in an endemic area of Nephropathia epidemica (NE) in Sweden. NE-patients monitored for 2 years had neutralizing antibodies in early sera collected 1-4 days after the onset of disease with a continuous increase in neutralizing antibodies with time. Furthermore, high titers of neutralizing antibodies were detected 10-20 years post-infection. This neutralization assay was also evaluated as a screening method in the production of monoclonal antibodies. The format of the NT-ELISA makes it feasible to screen a large number of specimens with results similar to the standard plaque or focus-reduction neutralization tests.

Synthesis and antiviral evaluation of N-carboxamidine-substituted analogues of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride.

Ten, hitherto unreported, analogues of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (2a, ribamidine) and methyl carboximidate 5 have been synthesized. These include the N-cyano (2b), N-alkyl (2c-e), N-amino acid (2f-h), N,N'-disubstituted (6, 7a,b), and the N-methylated carboxamide (1f) analogues of ribavirin. In addition, a new facile synthesis of carboxamidine 2a was also developed. All compounds were evaluated for biological activity against the following RNA viruses: Punta Toro (PT) and sandfly fever (SF) viruses (bunyaviruses); Japanese encephalitis (JE), yellow fever (YF), and dengue-4 viruses (flaviviruses); parainfluenza type 3 (PIV3), respiratory syncytial virus (RSV), and measles viruses (paramyxoviruses); influenza A and influenza B viruses (orthomyxoviruses); Venezuelan equine encephalomyelitis virus (VEE, alphavirus); human immunodeficiency virus type-1 (HIV-1, lentivirus); the DNA-containing vaccinia (VV) virus (poxvirus); and adeno type 5 (Ad5) viruses. All of the compounds except for 2b and 7a,b exhibited activity against the bunyaviruses such as that observed with 2a; however, higher IC50 values were generally observed. Glycine analogue 2f showed activity in PT-virus-infected mice in terms of increased survivors and decreased markers of viral pathogenicity. Carboxamidine 2a, carboximidate 5, and dimethyl amidine 6 exhibited activity against dengue type-4 virus. Monomethyl amidine 2c demonstrated activity against RSV, PIV3, and, to a lesser extent, influenza A and B. Activity of 2c generally required higher IC50 values than unsubstituted 2a. The latter exhibited hitherto unreported activity against RSV; therapeutic indices for 2a against RSV and PIV3 were greater than 64 and greater than 21. No substantial in vitro activity was observed for any of the compounds tested against Ad5, measles, JE, YF, VEE, or HIV-1. In addition, evidence is presented which argues in favor of a distinct antiviral mechanism of action for carboxamidines, e.g. 6, in contrast to a role as a carboxamide precursor.

Ribavirin mitigates wart growth in rabbits at early stages of infection with cottontail rabbit papillomavirus.

The challenge to develop antiviral agents effective against DNA viruses such as human papillomavirus (HPV) has been dependent on finding an animal model which mimics the human forms of the disease. We have used an existing model system for the purpose of measuring the effect of antiviral drugs on the inhibition of growth of these lesions. This was based upon domestic rabbits which efficiently grow cutaneous papillomas (warts) when infected with cottontail rabbit papillomavirus (CRPV). One agent which had shown significant success in achieving these goals was ribavirin. Ribavirin was administered intradermally shortly prior to infection at multiple sites with CRPV. Following daily injections of this drug for eight weeks, we have shown a dose-dependent response which had markedly reduced the number of warts, the time of first appearance of warts and reduced the tumor mass as compared to placebo-treated control animals. At the highest dose of ribavirin tested, 30 mg/kg/day, compared to controls, the average reduction in the number of warts was 52%, the average time of first appearance of warts was 49% longer, and the average mass of the warts was reduced by 98%. No detectable antibodies to CRPV were observed in any of the animals. The only side effects which were observed was focal alopecia, and a decrease in body growth upon prolonged treatment, both of which were completely reversible. Pharmacokinetic studies established the metabolism of ribavirin over a 24-h period of time. Ribavirin administered beginning 12 or 30 days post-infection, while not reducing the number of warts, slightly retarded the growth of warts as determined by date of first appearance of warts and mass of warts.

Prospective, double-blind, concurrent, placebo-controlled clinical trial of intravenous ribavirin therapy of hemorrhagic fever with renal syndrome.

A prospective, randomized, double-blind, concurrent, placebo-controlled clinical trial of intravenous ribavirin (loading dose of 33 mg/kg, 16 mg/kg every 6 h for 4 days, and 8 mg/kg every 8 h for 3 days) was conducted in 242 patients with serologically confirmed hemorrhagic fever with renal syndrome (HFRS) in the People's Republic of China. Mortality was significantly reduced (sevenfold decrease in risk) among ribavirin-treated patients, when comparisons were adjusted for baseline risk estimators of mortality (P = .01; two-tailed). HFRS typically consists of five consecutive but frequently overlapping clinical phases. Only occurrence of oliguric phase and hemorrhage was associated with severity of clinical disease in the placebo group. Ribavirin therapy also resulted in a significant reduction in the risk of entering the oliguric phase and experiencing hemorrhage. The only ribavirin-related side effect was a well-recognized, fully reversible anemia after completion of therapy.

Prophylactic and therapeutic activities of 7-thia-8-oxoguanosine against Punta Toro virus infections in mice.

The biological response modifier 7-thia-8-oxoguanosine was evaluated in mice against the hepatotropic Adames strain of Punta Toro virus. When administered intraperitoneally in divided doses, significant protection from death was conferred at doses of 50 and 100 mg/kg/day given 24 and 17 h pre-virus inoculation, 25-100 mg/kg/day administered 4 h pre- and 3 h post-virus challenge, and 12.5 to 100 mg/kg/day administered 24 and 31 h after virus inoculation. These doses preventing death reduced liver icterus scores, serum alanine aminotransferase and aspartate aminotransferase levels, and liver and serum virus titers relative to placebo controls. Full daily doses administered at 24 h were somewhat less protective to mice than divided daily doses starting at the same time. The initiation of treatment could be delayed as late as 36 h after virus inoculation, resulting in complete protection from mortality at 100 mg/kg/day. This prevention of death occurred despite the acute nature of the infection which resulted in deaths by 96 h in the placebo-treated controls. These results show that 7-thia-8-oxoguanosine has both prophylactic and therapeutic potential as an anti-Phlebovirus agent. Interferon induction appears to be the reason for antiviral activity in this model, since up to 10,000 units of interferon/ml were induced in mice 1 h after treatment with 100 mg 7-thia-8-oxoguanosine per kg, and antibody to interferon alpha/beta administered shortly after treatment with the nucleoside negated the antiviral effect.