Activity of a phenolic dibenzylsulfide against New World arenavirus infections.

BACKGROUND: Junín virus (JUNV) and several other clade B New World arenaviruses cause human disease ranging from mild febrile illness to severe viral haemorrhagic fever. These viruses pose a significant threat to national security and safe and effective therapies are limited except in Argentina, where immune plasma is the standard of care for treating JUNV infection in cases of Argentine haemorrhagic fever. METHODS: An in vitro screen of the Chemtura library identified several compounds with activity against Tacaribe virus (TCRV), a clade B arenavirus closely related to JUNV. Of these compounds, D746, a phenolic dibenzylsulfide, was further pursued for additional in vitro studies and evaluated in the AG129 mouse TCRV infection model. RESULTS: D746 was found to act during an early to intermediate stage of the TCRV replication cycle and µM range activity was confirmed by virus yield reduction assays with both TCRV and JUNV. Although intraperitoneal twice daily treatment regimens were found to be highly effective when started 2 h prior to TCRV challenge in AG129 mice, post-exposure treatment initiated 3 days after infection was not efficacious. Interestingly, despite the pre-exposure treatment success, D746 did not reduce serum or tissue virus titres during the acute infection. Moreover, D746 elicited ascites fluid accumulation in mice during, as well as independent of, infection. CONCLUSIONS: Our findings suggest that D746 may be altering the host response to TCRV infection in AG129 mice in a way that limits pathogenesis and thereby protects mice from otherwise lethal infection in the absence of measurable reductions in viral burden.

Effects of nasal or pulmonary delivered treatments with an adenovirus vectored interferon (mDEF201) on respiratory and systemic infections in mice caused by cowpox and vaccinia viruses.

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal cowpox (Brighton strain) and vaccinia (WR strain) virus respiratory and systemic infections in mice. Two routes of mDEF201 administration were used, nasal sinus (5-µl) and pulmonary (50-µl), to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. Lower respiratory infections (LRI), upper respiratory infections (URI), and systemic infections were induced by 50-µl intranasal, 10-µl intranasal, and 100-µl intraperitoneal virus challenges, respectively. mDEF201 treatments were given prophylactically either 24 h (short term) or 56d (long-term) prior to virus challenge. Single nasal sinus treatments of 10(6) and 10(7) PFU/mouse of mDEF201 protected all mice from vaccinia-induced LRI mortality (comparable to published studies with pulmonary delivered mDEF201). Systemic vaccinia infections responded significantly better to nasal sinus delivered mDEF201 than to pulmonary treatments. Cowpox LRI infections responded to 10(7) mDEF201 treatments, but a 10(6) dose was only weakly protective. Cowpox URI infections were equally treatable by nasal sinus and pulmonary delivered mDEF201 at 10(7) PFU/mouse. Dose-responsive prophylaxis with mDEF201, given one time only 56 d prior to initiating a vaccinia virus LRI infection, was 100% protective from 10(5) to 10(7) PFU/mouse. Improvements in lung hemorrhage score and lung weight were evident, as were decreases in liver, lung, and spleen virus titers. Thus, mDEF201 was able to treat different vaccinia and cowpox virus infections using both nasal sinus and pulmonary treatment regimens, supporting its development for humans.

Vascular leak ensues a vigorous proinflammatory cytokine response to Tacaribe arenavirus infection in AG129 mice.

BACKGROUND: Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV infection, but the pathogenic mechanisms contributing to the lethal disease are unclear. METHODS: To gain insights into the pathogenesis of TCRV infection in AG129 mice, we assessed hematologic and cytokine responses during the course of infection, as well as changes in the permeability of the vascular endothelium. We also treated TCRV-challenged mice with MY-24, a compound that prevents mortality without affecting viral loads during the acute infection, and measured serum and tissue viral titers out to 40 days post-infection to determine whether the virus is ultimately cleared in recovering mice. RESULTS: We found that the development of viremia and splenomegaly precedes an elevation in white blood cells and the detection of high levels of proinflammatory mediators known to destabilize the endothelial barrier, which likely contributes to the increased vascular permeability and weight loss that was observed several days prior to when the mice generally succumb to TCRV challenge. In surviving mice treated with MY-24, viremia and liver virus titers were not cleared until 2-3 weeks post-infection, after which the mice began to recover lost weight. Remarkably, substantial viral loads were still present in the lung, spleen, brain and kidney tissues at the conclusion of the study. CONCLUSIONS: Our findings suggest that vascular leak may be a contributing factor in the demise of TCRV-infected mice, as histopathologic findings are generally mild to moderate in nature, and as evidenced with MY-24 treatment, animals can survive in the face of high viral loads.

Extended protection against phlebovirus infection conferred by recombinant adenovirus expressing consensus interferon (DEF201).

Punta Toro virus (PTV; Bunyaviridae, Phlebovirus) is related to Rift Valley fever virus (RVFV), a pathogenic agent which causes severe disease in humans and livestock primarily in the sub-Saharan region of Africa. The recent range expansion of RVFV and the potential for its intentional release into naïve populations pose a significant threat to public health and agriculture. Studies modeling disease in rodents and nonhuman primates have shown that PTV and RVFV are highly sensitive to the antiviral effects of alpha interferon (IFN-α), an important component of the innate antiviral host response. While recombinant IFN-α has high therapeutic value, its utility for the treatment of neglected tropical diseases is hindered by its short in vivo half-life and costly production of longer-lasting pegylated IFNs. Here, we demonstrate extended preexposure protection against lethal PTV challenge following a single intranasal administration of DEF201, which is a replication-deficient human adenovirus type 5 vector engineered to constitutively express consensus IFN-α (cIFN-α) from transduced host cells. DEF201 was also efficacious when administered within 24 h as a postexposure countermeasure. Serum concentrations of cIFN-α could be detected as early as 8 h following treatment and persisted for more than 1 week. The prolonged antiphlebovirus prophylactic effect, low production costs, and ease of administration make DEF201 a promising agent for intervention during natural disease outbreaks and for countering possible bioterrorist acts.

Use of recombinant adenovirus vectored consensus IFN-α to avert severe arenavirus infection.

Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.

Therapy and long-term prophylaxis of vaccinia virus respiratory infections in mice with an adenovirus-vectored interferon alpha (mDEF201).

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal vaccinia virus (WR strain) respiratory infections in mice. mDEF201 was administered as a single intranasal treatment either prophylactically or therapeutically at doses of 10(6) to 10(8) plaque forming units/mouse. When the prophylactic treatment was given at 56 days prior to infection, it protected 90% of animals from death (100% protection for treatments given between 1-49 days pre-infection), with minimal weight loss occurring during infection. Surviving animals re-challenged with virus 22 days after the primary infection were protected from death, indicating that mDEF201 did not compromise the immune response against the initial infection. Post-exposure therapy was given between 6-24 h after vaccinia virus exposure and protection was afforded by a 10(8) dose of mDEF201 given at 24 h, whereas a 10(7) dose was effective up to 12 h. Comparisons were made of the ability of mDEF201, given either 28 or 1 day prior to infection, to inhibit tissue virus titers and lung infection parameters. Lung, liver, and spleen virus titers were inhibited to nearly the same extent by either treatment, as were lung weights and lung hemorrhage scores (indicators of pneumonitis). Lung virus titers were significantly (>100-fold) lower than in the placebo group, and the other infection parameters in mDEF201 treated mice were nearly at baseline. In contrast, viral titers and lung infection parameters were high in the placebo group on day 5 of the infection. These results demonstrate the long-acting prophylactic and treatment capacity of mDEF201 to combat vaccinia virus infections.

Effects of TheraMax on influenza virus infections in cell culture and in mice.

BACKGROUND: Limited in vivo studies in the scientific literature suggest that components of green tea and elderberry may be beneficial in treating influenza virus infections. They are thought to act by blocking virus adsorption to cells. TheraMax(®) is a proprietary medication administered by nasal spray that contains both green tea and elderberry extracts that was evaluated for antiviral activity. METHODS: TheraMax was tested by dilution in Madin-Darby canine kidney cell cultures in standard viral cytopathic effect (CPE) inhibition and virucidal assays against eight influenza A and B strains. It was also administered intranasally to mice to determine protective activity compared to oral oseltamivir against an influenza A/NWS/33 (H1N1) infection. RESULTS: In cell culture assays, TheraMax was found to inhibit viral CPE by 50% at a 1:20 dilution for seven of the eight virus strains, with no virucidal activity at 1:2 dilution. The undiluted product was administered to anaesthetized mice twice daily for 4 days starting 2 h before or 12 h after infection. Alternatively, TheraMax and virus were combined for treatment and infection. Oseltamivir was given orally twice daily for 5 days at 10 mg/kg/day starting at -2 h. TheraMax (combined directly with virus) and oseltamivir each prevented death and curtailed weight loss during the infection, and improved lung haemorrhage scores on day 6. TheraMax starting at -2 h or 12 h significantly delayed death by >2 days and reduced lung haemorrhage scores, but most animals died. CONCLUSIONS: These combined results indicate that TheraMax delayed symptoms during animal infections, likely through blocking of virus adsorption.

Topical treatment of cutaneous vaccinia virus infections in immunosuppressed hairless mice with selected antiviral substances.

BACKGROUND: Certain nucleoside, nucleotide and pyrophosphate analogues may be useful for treating severe complications arising as a result of virus dissemination following smallpox (live vaccinia virus) vaccinations, especially in immunocompromised individuals. We used an immunosuppressed hairless mouse model to study the effects of 10 antiviral agents on progressive vaccinia infections. METHODS: Hairless mice were immunosuppressed by treatment with cyclophosphamide (100 mg/kg) every 4 days starting 1 day prior to vaccinia virus (WR strain) infection of wounded skin. Topical treatments with antiviral agents were applied twice a day for 7 days starting 5 days after virus exposure. RESULTS: Topical 1% cidofovir cream treatment was effective in significantly reducing primary lesion severity and decreasing the number of satellite lesions. Topical 1% cyclic HPMPC and 1% phosphonoacetic acid were not quite as active as cidofovir. Ribavirin (5%) treatment reduced lesion severity and diminished the numbers of satellite lesions, but the mice died significantly sooner than placebos. 2-Amino-7-[(1,3,-dihydroxy-2-propoxy)methyl]purine (compound S2242; 1%) moderately reduced primary lesion sizes. Ineffective treatments included 5% arabinosyladenine, 1% arabinosylcytosine, 1% 5-chloro-arabinosylcytosine, 5% arabinosylhypoxanthine 5-monophosphate and 5% viramidine. CONCLUSIONS: Of the compounds tested, topically applied cidofovir was the most effective treatment of cutaneous vaccinia virus infections in immunosuppressed mice. Topical treatment with cidofovir could be considered as an adjunct to intravenous drug therapy for serious infections.

Evaluation of imiquimod for topical treatment of vaccinia virus cutaneous infections in immunosuppressed hairless mice.

Imiquimod is an immune response modifier prescribed as a topical medication for a number of viral and neoplastic conditions. We evaluated the antiviral activity of imiquimod against vaccinia virus (WR strain) cutaneous infections in immunosuppressed (with cyclophosphamide) hairless mice when administered after virus exposure. Primary lesions progressed in severity, satellite lesions developed, and infection eventually killed the mice. Once daily topical treatment with 1% imiquimod cream for 3, 4, or 5 days were compared to twice daily topical treatment with 1% cidofovir cream for 7 days. Survival time of mice in all treated groups was significantly prolonged compared to placebo controls. The mean day of death for the placebo group, 3-day imiquimod, 4-day imiquimod, 5-day imiquimod, and cidofovir groups were 15.5, 20.0, 20.5, 19.5, and 20.5 days post-infection, respectively. All treatment groups showed significant reductions in primary lesion size and in the number of satellite lesions. The cidofovir and 4-day imiquimod treatments delayed the appearance of lung virus titers by 3 and 6 days, respectively, although cutaneous lesion and snout virus titers were not as affected by treatment. Benefits in survival and lesion reduction were observed when imiquimod treatment was delayed from 24, 48, and 72 h post-infection. However, increasing the treatment dose of imiquimod from 1% to 5% led to a significant decrease in antiviral efficacy. These results demonstrate the protective effects of topically administered imiquimod against a disseminated vaccinia virus infection in this mouse model.

Development of a new tacaribe arenavirus infection model and its use to explore antiviral activity of a novel aristeromycin analog.

BACKGROUND: A growing number of arenaviruses can cause a devastating viral hemorrhagic fever (VHF) syndrome. They pose a public health threat as emerging viruses and because of their potential use as bioterror agents. All of the highly pathogenic New World arenaviruses (NWA) phylogenetically segregate into clade B and require maximum biosafety containment facilities for their study. Tacaribe virus (TCRV) is a nonpathogenic member of clade B that is closely related to the VHF arenaviruses at the amino acid level. Despite this relatedness, TCRV lacks the ability to antagonize the host interferon (IFN) response, which likely contributes to its inability to cause disease in animals other than newborn mice. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a new mouse model based on TCRV challenge of AG129 IFN-α/ß and -γ receptor-deficient mice. Titration of the virus by intraperitoneal (i.p.) challenge of AG129 mice resulted in an LD(50) of ∼100 fifty percent cell culture infectious doses. Virus replication was evident in the serum, liver, lung, spleen, and brain 4-8 days after inoculation. MY-24, an aristeromycin derivative active against TCRV in cell culture at 0.9 µM, administered i.p. once daily for 7 days, offered highly significant (P<0.001) protection against mortality in the AG129 mouse TCRV infection model, without appreciably reducing viral burden. In contrast, in a hamster model of arenaviral hemorrhagic fever based on challenge with clade A Pichinde arenavirus, MY-24 did not offer significant protection against mortality. CONCLUSIONS/SIGNIFICANCE: MY-24 is believed to act as an inhibitor of S-adenosyl-L-homocysteine hydrolase, but our findings suggest that it may ameliorate disease by blunting the effects of the host response that play a role in disease pathogenesis. The new AG129 mouse TCRV infection model provides a safe and cost-effective means to conduct early-stage pre-clinical evaluations of candidate antiviral therapies that target clade B arenaviruses.

Assessing changes in vascular permeability in a hamster model of viral hemorrhagic fever.

BACKGROUND: A number of RNA viruses cause viral hemorrhagic fever (VHF), in which proinflammatory mediators released from infected cells induce increased permeability of the endothelial lining of blood vessels, leading to loss of plasma volume, hypotension, multi-organ failure, shock and death. The optimal treatment of VHF should therefore include both the use of antiviral drugs to inhibit viral replication and measures to prevent or correct changes in vascular function. Although rodent models have been used to evaluate treatments for increased vascular permeability (VP) in bacterial sepsis, such studies have not been performed for VHF. RESULTS: Here, we use an established model of Pichinde virus infection of hamsters to demonstrate how changes in VP can be detected by intravenous infusion of Evans blue dye (EBD), and compare those measurements to changes in hematocrit, serum albumin concentration and serum levels of proinflammatory mediators. We show that EBD injected into sick animals in the late stage of infection is rapidly sequestered in the viscera, while in healthy animals it remains within the plasma, causing the skin to turn a marked blue color. This test could be used in live animals to detect increased VP and to assess the ability of antiviral drugs and vasoactive compounds to prevent its onset. Finally, we describe a multiplexed assay to measure levels of serum factors during the course of Pichinde arenavirus infection and demonstrate that viremia and subsequent increase in white blood cell counts precede the elaboration of inflammatory mediators, which is followed by increased VP and death. CONCLUSIONS: This level of model characterization is essential to the evaluation of novel interventions designed to control the effects of virus-induced hypercytokinemia on host vascular function in VHF, which could lead to improved survival.

Combinations of oseltamivir and peramivir for the treatment of influenza A (H1N1) virus infections in cell culture and in mice.

Oseltamivir and peramivir are being considered for combination treatment of serious influenza virus infections in humans. Both compounds are influenza virus neuraminidase inhibitors, and since peramivir binds tighter to the enzyme than oseltamivir carboxylate (the active form of oseltamivir), the possibility exists that antagonistic interactions might result when using the two compounds together. To study this possibility, combination chemotherapy experiments were conducted in vitro and in mice infected with influenza A/NWS/33 (H1N1) virus. Treatment of infected MDCK cells was performed with combinations of oseltamivir carboxylate and peramivir at 0.32-100µM for 3 days, followed by virus yield determinations. Additive drug interactions with a narrow region of synergy were found using the MacSynergy method. In a viral neuraminidase assay with combinations of inhibitors at 0.01-10nM, no significant antagonistic or synergistic interactions were observed across the range of concentrations. Infected mice were treated twice daily for 5 days starting 2h prior to virus challenge using drug doses of 0.05-0.4mg/kg/day. Consistent and statistically significant increases in the numbers of survivors were seen when twice daily oral oseltamivir (0.4mg/kg/day) was combined with twice daily intramuscular peramivir (0.1 and 0.2mg/kg/day) compared to single drug treatments. The data demonstrate that combinations of oseltamivir and peramivir perform better than suboptimal doses of each compound alone to treat influenza infections in mice. Treatment with these two compounds should be considered as an option.

Lack of efficacy of aurintricarboxylic acid and ethacrynic acid against vaccinia virus respiratory infections in mice.

BACKGROUND: Aurintricarboxylic acid (ATA) and ethacrynic acid (ECA) have been reported to exhibit antiviral activity against vaccinia virus infections in cell culture by inhibiting early and late gene transcription, respectively. The purpose of this work was to determine if these inhibitors would effectively treat vaccinia virus infections in mice, which has not previously been studied. METHODS: ECA was investigated by cell culture plaque reduction assay for the inhibition of cowpox and vaccinia virus infections to clarify issues regarding its potency and selectivity. Mice infected intranasally with vaccinia virus were treated by intraperitoneal route twice daily for 5 days with ATA (10 and 30 mg/kg/day) and ECA (15 and 30 mg/kg/day) or once daily for 2 days with cidofovir (100 mg/kg/day). RESULTS: ECA caused 50% inhibition of virus plaque formation at 20-79 muM in four cultured cell lines, with 50% cytotoxicity at 84-173 muM, giving low (1.3-4.2) selectivity index values. Preliminary toxicity tests in uninfected mice indicated that ATA and ECA were both overtly toxic at 100 mg/kg/day. No protection from mortality was afforded by treatment of vaccinia virus infections with ATA or ECA, but 100% survival was achieved in the cidofovir group. ATA- and ECA-treated mice died significantly sooner than placebo-treated animals, indicating that these compounds exacerbated the infection. CONCLUSIONS: Both ATA and ECA lack antiviral potency and selectivity in cell culture. The compounds were ineffective in treating mice at intraperitoneal doses of

Induction of interferon-gamma-inducible protein 10 by SARS-CoV infection, interferon alfacon 1 and interferon inducer in human bronchial epithelial Calu-3 cells and BALB/c mice.

BACKGROUND: The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is poorly understood. Several mechanisms involving both direct effects on target cells and indirect effects via the immune system might exist. SARS-CoV has been shown in vitro to induce changes of cytokines and chemokines in various human and animal cells. We previously reported that interferon (IFN) alfacon-1 was more active against SARS-CoV infection in human bronchial epithelial Calu-3 cells than in African green monkey kidney epithelial cells on day 3 post-infection. METHODS: In the current study, we first evaluated the efficacy of IFN-alfacon 1 in Calu-3 cells during the first 7 days of virus infection. We then used the two-antibody sandwich ELISA method to detect IFN-gamma-inducible protein 10 (IP-10). We further evaluated the efficacy of antivirals directed against SARS-CoV infection in BALB/c mice. RESULTS: A potent, prolonged inhibition of SARS-CoV replication in Calu-3 cells with IFN-alfacon 1 was observed. Furthermore, IP-10, an IFN-inducible leukocyte chemoattractant, was detected in Calu-3 cells after SARS-CoV infection. Interestingly, IP-10 expression was shown to be significantly increased when SARS-CoV-infected Calu-3 cells were treated with IFN alfacon-1. IP-10 expression was detected in the lungs of SARS-CoV-infected BALB/c mice. Significantly high levels of mouse IP-10 in BALB/c mice was also detected when SARS-CoV-infected mice were treated with the interferon inducer, polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose (poly IC:LC). Treatment with poly IC:LC by intranasal route were effective in protecting mice against a lethal infection with mouse-adapted SARS-CoV and reduced the viral lung titres. CONCLUSIONS: Our data might provide an important insight into the mechanism of pathogenesis of SARS-CoV and these properties might be therapeutically advantageous.

Efficacy of favipiravir (T-705) and T-1106 pyrazine derivatives in phlebovirus disease models.

Several studies have reported favipiravir (T-705) to be effective in treating a number of viral diseases modeled in rodent systems. Notably, the related pyrazine derivative, T-1106, was found to be more effective than T-705 in treating yellow fever virus infection in hamsters. Based on these findings, we hypothesized that T-1106 may be more effective in treating hepatotropic Punta Toro virus (PTV, Phlebovirus) infection in rodents. In cell culture, the inhibitory concentrations of the compounds against various phleboviruses ranged from 3 to 55microM for T-705 and from 76 to 743microM for T-1106. In PTV-challenged hamsters, a model that generally presents with high liver viral loads, T-1106 was more effective at reducing mortality. However, in mice infected with PTV, a model wherein systemic infection is more prominent, the greater efficacy exhibited by T-1106 in the hamster system was not apparent. In contrast, T-705 was superior in preventing mortality in hamsters challenged with Pichinde virus (PICV, Arenavirus), an infection characterized as diffuse and pantropic. Remarkably, T-1106 has proven more active in vivo than would have been expected from our cell culture results, and our in vivo findings suggest that it is more effective in infections characterized predominantly by high levels of hepatic viral burden.

Effects of the combination of favipiravir (T-705) and oseltamivir on influenza A virus infections in mice.

Favipiravir (T-705 [6-fluoro-3-hydroxy-2-pyrazinecarboxamide]) and oseltamivir were combined to treat influenza virus A/NWS/33 (H1N1), A/Victoria/3/75 (H3N2), and A/Duck/MN/1525/81 (H5N1) infections. T-705 alone inhibited viruses in cell culture at 1.4 to 4.3 microM. Oseltamivir inhibited these three viruses in cells at 3.7, 0.02, and 0.16 microM and in neuraminidase assays at 0.94, 0.46, and 2.31 nM, respectively. Oral treatments were given twice daily to mice for 5 to 7 days starting, generally, 24 h after infection. Survival resulting from 5 days of oseltamivir treatment (0.1 and 0.3 mg/kg/day) was significantly better in combination with 20 mg/kg of body weight/day of T-705 against the H1N1 infection. Treatment of the H3N2 infection required 50 mg/kg/day of oseltamivir for 7 days to achieve 60% protection; 25 mg/kg/day was ineffective. T-705 was >or=70% protective at 50 to 100 mg/kg/day but inactive at 25 mg/kg/day. The combination of inhibitors (25 mg/kg/day each) increased survival to 90%. The H5N1 infection was not benefited by treatment with oseltamivir (

Punta Toro virus (Bunyaviridae, Phlebovirus) infection in mice: strain differences in pathogenesis and host interferon response.

The Adames strain of Punta Toro virus (PTV-A, Bunyaviridae, Phlebovirus) causes an acute lethal disease in hamsters and mice. The Balliet strain of the virus (PTV-B) is generally considered to be avirulent. The difference in hamster susceptibility is likely due to the ability of PTV-A to suppress interferon (IFN)-beta similarly to that described for Rift Valley fever virus. Here we investigated strain differences in PTV pathogenesis and the IFN response in mice. Although PTV-B infection in mice did not induce systemic IFN-beta release, primary macrophages produced dramatically higher levels when exposed to the virus in culture. The importance of IFN in resistance to PTV infection was borne out in studies employing STAT-1 knock-out mice. Also, a number of genes specific to IFN response pathways were upregulated in PTV-B-infected macrophages. Our findings provide new insights into the type I IFN response during PTV infection in the mouse model of phleboviral disease.

Effects of double combinations of amantadine, oseltamivir, and ribavirin on influenza A (H5N1) virus infections in cell culture and in mice.

An amantadine-resistant influenza A/Duck/MN/1525/81 (H5N1) virus was developed from the low-pathogenic North American wild-type (amantadine-sensitive) virus for studying treatment of infections in cell culture and in mice. Double combinations of amantadine, oseltamivir (or the cell culture-active form, oseltamivir carboxylate), and ribavirin were used. Amantadine-oseltamivir carboxylate and amantadine-ribavirin combinations showed synergistic interactions over a range of doses against wild-type virus in Madin-Darby canine kidney (MDCK) cell culture, but oseltamivir carboxylate-ribavirin combinations did not. Primarily additive interactions were seen with oseltamivir carboxylate-ribavirin combinations against amantadine-resistant virus. The presence of amantadine in drug combinations against the resistant virus did not improve activity. The wild-type and amantadine-resistant viruses were lethal to mice by intranasal instillation. The resistant virus infection could not be treated with amantadine up to 100 mg/kg body weight/day, whereas the wild-type virus infection was treatable with oral doses of 10 (weakly effective) to 100 mg/kg/day administered twice a day for 5 days starting 4 h prior to virus exposure. Drug combination studies showed that treatment of the amantadine-resistant virus infection with amantadine-oseltamivir or amantadine-ribavirin combinations was not significantly better than using oseltamivir or ribavirin alone. In contrast, the oseltamivir-ribavirin (25- and 75-mg/kg/day combination) treatments produced significant reductions in mortality. The wild-type virus infection was markedly reduced in severity by all three combinations (amantadine, 10 mg/kg/day combined with the other compounds at 20 or 40 mg/kg/day) compared to monotherapy with the three compounds. Results indicate a lack of benefit of amantadine in combinations against amantadine-resistant virus, but positive benefits in combinations against amantadine-sensitive virus.

Prophylaxis with cationic liposome-DNA complexes protects hamsters from phleboviral disease: importance of liposomal delivery and CpG motifs.

Cationic liposome-DNA complexes (CLDC) are cationic/neutral lipid carriers complexed with plasmid DNA that when administered systemically results in a robust T(H)1 cytokine response. CLDC have been shown to be effective in prophylaxis and therapeutic treatment of animal models of viral disease. To determine the contribution of liposomal delivery and CpG content of the plasmid DNA to the efficacy of CLDC; plasmid, CpG-free plasmid DNA, or CpG-containing oligodeoxynucleotides (ODN) with and without liposomes, as well as poly(I:C(12)U), were evaluated for their ability to elicit protection against lethal Punta Toro virus (PTV, Bunyaviridae, phlebovirus) challenge in hamsters. CLDC-containing plasmid significantly improved survival, decreased systemic and liver viral loads, and reduced liver damage due to progression of viral infection. Mouse-reactive ODNs complexed with liposomes failed to protect hamsters, whereas ODNs known to cross-react with human and mouse (CpG 2006) or non-liposomal poly(I:C(12)U) showed survival benefit but did not limit liver injury. Liposomes complexed with a non-CpG motif-containing plasmid reduced liver viral load and tissue damage, but did not protect hamsters from death. To evaluate the mechanisms of the enhanced activity of CLDC, microarray experiments examined differences in the gene expression profile. The results suggest a broad T(H)1 response elicited by liposomal delivery of a diverse sequence containing CpG and non-CpG elements may be a more effective antiviral treatment than other nucleic acid based immunotherapeutics.

Treatment of late stage disease in a model of arenaviral hemorrhagic fever: T-705 efficacy and reduced toxicity suggests an alternative to ribavirin.

A growing number of arenaviruses are known to cause viral hemorrhagic fever (HF), a severe and life-threatening syndrome characterized by fever, malaise, and increased vascular permeability. Ribavirin, the only licensed antiviral indicated for the treatment of certain arenaviral HFs, has had mixed success and significant toxicity. Since severe arenaviral infections initially do not present with distinguishing symptoms and are difficult to clinically diagnose at early stages, it is of utmost importance to identify antiviral therapies effective at later stages of infection. We have previously reported that T-705, a substituted pyrazine derivative currently under development as an anti-influenza drug, is highly active in hamsters infected with Pichinde virus when the drug is administered orally early during the course of infection. Here we demonstrate that T-705 offers significant protection against this lethal arenaviral infection in hamsters when treatment is begun after the animals are ill and the day before the animals begin to succumb to disease. Importantly, this coincides with the time when peak viral loads are present in most organs and considerable tissue damage is evident. We also show that T-705 is as effective as, and less toxic than, ribavirin, as infected T-705-treated hamsters on average maintain their weight better and recover more rapidly than animals treated with ribavirin. Further, there was no added benefit to combination therapy with T-705 and ribavirin. Finally, pharmacokinetic data indicate that plasma T-705 levels following oral administration are markedly reduced during the latter stages of disease, and may contribute to the reduced efficacy seen when treatment is withheld until day 7 of infection. Our findings support further pre-clinical development of T-705 for the treatment of severe arenaviral infections.