Justin G. Julander

Defining Limits of Treatment with Humanized Neutralizing Monoclonal Antibody for West Nile Virus Neurological Infection in a Hamster Model

John D. Morrey et al. — Thu, 15 Dec 2011 13:24:37 PST

A potent anti-West Nile virus (anti-WNV)-neutralizing humanized monoclonal antibody, hE16, was previously shown to improve the survival of WNV-infected hamsters when it was administered intraperitoneally (i.p.), even after the virus had infected neurons in the brain. In this study, we evaluated the therapeutic limit of hE16 for the treatment of WNV infection in hamsters by comparing single-dose peripheral (i.p.) therapy with direct administration into the pons through a convection-enhanced delivery (CED) system. At day 5 after infection, treatments with hE16 by the peripheral and the CED routes were equally effective at reducing morbidity and mortality. In contrast, at day 6 only the treatment by the CED route protected the hamsters from lethal infection. These experiments suggest that hE16 can directly control WNV infection in the central nervous system. In support of this, hE16 administered i.p. was detected in a time-dependent manner in the serum, cerebrospinal fluid (CSF), cerebral cortex, brain stem, and spinal cord in CSF. A linear relationship between the hE16 dose and the concentration in serum was observed, and maximal therapeutic activity occurred at doses of 0.32 mg/kg of body weight or higher, which produced serum hE16 concentrations of 1.3 µg/ml or higher. Overall, these data suggest that in hamsters hE16 can ameliorate neurological disease after significant viral replication has occurred, although there is a time window that limits therapeutic efficacy.

Treatment of Venezuelan Equine Encephalitis Virus Infection with (-)-Carbodine

Justin G. Julander et al. — Thu, 15 Dec 2011 13:24:36 PST

Venezuelan equine encephalitis virus (VEEV) may cause encephalitis in humans, for which no FDA-approved antiviral treatment is available. Carbocyclic cytosine (carbodine) has broad-spectrum activity but toxicity has limited its utility. It was anticipated that one of the enantiomers of carbodine would show enhanced activity and reduced toxicity. The activity of the d-(−) enantiomer of carbodine [(−)-carbodine] was evaluated by infectious cell culture assay and was found to have a 50% effective concentration (EC50) of 0.2 μg/ml against the TC-83 vaccine strain of VEEV in Vero cells, while the l-(+) enantiomer had no activity. Virus titer inhibition correlated with intracellular cytidine triphosphate reduction after treatment with (−)-carbodine, as determined by HPLC analysis. Pre-treatment with 200 mg/(kg d) resulted in significant improvement in survival, virus load in the brain, weight change, and mean day-to-death in a mouse model of TC-83 VEEV disease. A single dose of (−)-carbodine resulted in a slight extension of mean time to death in mice infected with wild-type VEEV. Post-virus exposure treatment with (−)-carbodine was effective in significantly improving disease parameters in mice infected with TC-83 VEEV when treatment was initiated as late as 4 days post-virus installation (dpi). It is remarkable that (−)-carbodine is effective when initiated after the establishment of brain infection.

Effect of Interferon-Alpha and Interferon- Inducers on West Nile Virus in Mouse and Hamster Animal Models

John D. Morrey et al. — Thu, 15 Dec 2011 13:24:34 PST

The recent West Nile virus (WNV) outbreak in the United States has increased the need to identify effective therapies. Studies were conducted in cell culture and in rodent animal models to determine the efficacy of interferon-alpha (IFN-α), interferon (IFN) inducers and ribavirin, alone or in combination with IFN, in treating WNV. Intraperitoneal injection of IFN-α B/D (qd for 7 days), polyI-polyC(12)U [Ampligen (every other day for 7 days)] and topically applied imiquimod (qd for 7 days), administered from 1 day before viral challenge, were effective in protecting, respectively, 100%, 100% and 70% of BALB/c mice from mortality induced by subcutaneous injection of WNV. When IFN-α B/D or Ampligen treatments were delayed to 4–6 h before viral challenge in mice, efficacy was greatly diminished. Infected Syrian golden hamsters treated with interferon alphacon-1 (Infergen) and Ampligen 4–6 h before viral challenge gained more weight and had a greater survival than saline-treated animals. A combination study of subcutaneously administered Infergen (5 to 0.05 µg/kg/day) and ribavirin (75 to 7.5 mg/kg/day) in >7 week old hamsters demonstrated that Infergen was slightly efficacious in reducing mortality and disease signs; however, it was not synergistic in its antiviral effects when combined with ribavirin. Ribavirin treatment alone increased mortality of infected hamsters. The reduced mortality correlated with reduced plasma viraemia. Since WNV-infected patients have already been treated with IFN and ribavirin and future clinical trials have been suggested, this first report of IFN alone or in combination with ribavirin in WNV-infected animal models might provide useful information for subsequent treatment of patients.

Activity of T-1106 in a Hamster Model of Yellow Fever Virus Infection

Justin G. Julander et al. — Thu, 15 Dec 2011 13:24:32 PST

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.

West Nile Virus Infection of the Placenta

Justin G. Julander et al. — Thu, 15 Dec 2011 13:24:31 PST

Intrauterine infection of fetuses with West Nile virus (WNV) has been implicated in cases of women infected during pregnancy. Infection of timed-pregnant mice on 5.5, 7.5, and 9.5 days post-coitus (dpc) resulted in fetal infection. Infection of dams on 11.5 and 14.5 dpc resulted in little and no fetal infection, respectively. Pre-implantation embryos in culture were also infected with WNV after the blastocyst stage and the formation of trophectoderm. Green fluorescent protein (GFP) expression was observed in a trophoblast stem (TS) cell line after infection with a GFP-expressing WNV construct. However, no fluorescence was observed in differentiated trophoblast giant cell (TGC) cultures. GFP fluorescence was present in TGC cultures if infected TS cells were induced to differentiate. These results suggest that embryos are susceptible to WNV infection after the formation of the trophectoderm around 3.5 dpc through the formation of the functional placenta around 10.5 dpc.

Prophylactic Treatment with Recombinant Eimeria Protein, Alone or in Combination with an Agonist Cocktail, Protects Mice from Banzi Virus Infection

Justin G. Julander et al. — Thu, 15 Dec 2011 13:24:29 PST

A recombinant Eimeria protozoan protein antigen (rEA) has been shown to have antitumor and antiviral activity. The purpose of this study was to determine the effect of rEA treatment alone or in combination with an agonist cocktail consisting of granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-γ), interleukin 4 (IL-4), and anti CD-40 antibody, in the treatment of Banzi virus (BV) disease in BALB/c mice. Treatment with rEA resulted in a significant increase in survival, weight gain, and mean day to death in BV-infected mice and resulted in a significant decrease in brain virus titer. Treatment with rEA, in combination with a 4-agonist cocktail, improved disease parameters to a greater degree than rEA treatment alone. The effect of treatment with a reduced concentration of agonist cocktail or fewer components of the agonist cocktail, in combination with rEA, on disease outcome in BV-infected mice was also investigated. Treatment with rEA, alone or in combination with agonist cocktail, 24 h after virus challenge did not improve disease. Treatment with rEA, alone or in combination with an agonist cocktail, is efficacious for the prophylaxis of BV infection in mice.

Novel 3-Sulphonamido-Quinazolin-4(3H)-One Derivatives: Microwave-Assisted Synthesis and Evaluation of Antiviral Activities against Respiratory and Biodefense Viruses

P. Selvam et al. — Thu, 15 Dec 2011 13:24:27 PST

We designed and synthesized novel 2,3-disubstituted quinazolin-4(3H)-ones by microwave technique and characterized them by spectral analysis. Synthesized compounds were screened for cytotoxicity and for antiviral activity against influenza A (H1N1, H3N2 and H5N1), severe acute respiratory syndrome corona, dengue, yellow fever, Venezuelan equine encephalitis (VEE), Rift Valley fever, and Tacaribe viruses in cell culture. A neutral red uptake assay was used to determine 50% virus-inhibitory concentrations (EC50) of test compounds and their 50% cytotoxicity concentration (CC50) in uninfected Madin–Darby canine kidney, Vero, and Vero 76 cells; selectivity indices (ratio of CC50 to EC50) were derived from the data. The compound 4-(6,8-dibromo-4-oxo-2-phenyl quinazolin-3(4H)-yl)-N-(4,5-dimethyloxazol-2yl) benzenesulphonamide 15 inhibited the replication of avian influenza (H5N1) virus (EC50=8.4 µg/ml, CC50>100 µg/ml, SI>11.9) as did 4-(6-bromo-4oxo-2phenylquinazolin-3(4H)-yl) benzene] sulphonamide 5 (EC50=3 µg/ml, CC50= 32 µg/ml, SI=11). Compound 5 was also moderately active against VEE and Tacaribe viruses. The methodology described in this report is applicable for rapid synthesis of many compounds with potential antiviral properties.

Modeling Hamsters for Evaluating West Nile Virus Therapies

John D. Morrey et al. — Thu, 15 Dec 2011 13:24:26 PST

A hamster model infected with a New York crow brain isolate of West Nile virus (WNV) was characterized for evaluating potential antiviral therapies. Older hamsters (7–11 weeks old) had a lower mortality of not, vert, similar50% and more apparent disease signs as compared to >90% mortality in younger hamsters and mice. Disease signs such as limb strength, lacrimation, front limb tremors, somnolence, and deficiences in neurological responses were noted at different times after viral injection. Weight loss was a marker for WNV disease signs, whereas, the ability to climb up an inclined ramp was associated with whether the animals survived the disease or died. Infectious WNV assays performed on tissues from hamsters during development of the infection indicated that viral titers peaked first in plasma, but that titers were eventually highest in kidney tissue. Viral titers achieved maximal levels in brain tissue on 6 dpi, which was 1–2 days before strong neurological signs and death started to occur. Maximal spleen and plasma titers were achieved sooner in young hamsters as compared with older hamsters, which correlated with increased susceptibility. To test the hypothesis that older hamsters would be more sensitive for identifying antiviral effects, Infergen, a consensus human interferon-α highly active against WNV in cell culture, was administered subcutaneously to older and younger hamsters beginning on 2 dpi. The effects of Infergen on weight change, survival, and climbing ability of infected animals were more apparent in older hamsters than in younger hamsters. The use of older hamsters is another WNV-infectious model, in addition to mice, for evaluating potential antiviral therapies.

Anti-Hepatitis B Virus Activity of ORI-9020, a Novel Phosphorothioate Dinucleotide, in a Transgenic Mouse Model

R. P. Iyer et al. — Thu, 15 Dec 2011 13:24:24 PST

ORI-9020, a novel dinucleotide, evaluated in transgenic mice expressing hepatitis B virus (HBV), significantly reduced liver HBV DNA (P <= 0.001). Levels of HBeAg and HBsAg in serum and of HBcAg in liver were not affected by treatment. A minimal effective dosage was determined to be between 1.6 and 0.5 mg/kg of body weight/day, which was similar to that observed for adefovir dipivoxil.

Comparison of the Inhibitory Effects of Interferon Alfacon-1 and Ribavirin on Yellow Fever Virus Infection in a Hamster Model

Justin G. Julander et al. — Thu, 15 Dec 2011 13:24:23 PST

Antiviral compounds were evaluated for efficacy against yellow fever virus (YFV) in a hamster model of YFV-induced liver disease. Challenge with a 10^2 50% cell culture infectious doses of YFV resulted in a 50–80% mortality rate in female hamsters. Virus was detected by quantitative real-time RT-PCR (QRT-PCR) in liver, kidney, spleen and serum with peak titers on 4–6 days post-viral challenge (dpi). Serum levels of alkaline phosphatase, alanine aminotransferase (ALT), bilirubin, blood urea nitrogen, potassium and creatinine were significantly elevated, while serum levels of albumin, amylase, glucose, calcium, globulin, phosphorus, sodium and total protein were significantly reduced. Packed cell volume and white blood cell count were significantly elevated during the course of the infection. Intraperitoneal treatment of hamsters with 0.5–5 μg/kg/day interferon (IFN) alfacon-1, 100 mg/kg/day viramidine or 50 mg/kg/day ribavirin, initiated 4 h prior to YFV challenge, resulted in significant improvement in survival and serum ALT levels. Treatment with IFN alfacon-1 or ribavirin starting 2 dpi, also significantly improved survival and serum ALT levels in hamsters challenged with YFV. Pre- and post-virus exposure treatment with IFN alfacon-1 was efficacious in improving disease in YFV-infected hamsters.

Effect of Exogenous Interferon and an Interferon Inducer on Western Equine Encephalitis Virus Disease in a Hamster Model

Justin G. Julander et al. — Thu, 15 Dec 2011 13:24:21 PST

Mice are used as models for western equine encephalitis virus (WEEV) infection, but high mortality is generally only seen with intracranial or intranasal challenge, while peripheral inoculation results in approximately 50% mortality and is not dose-dependent. Hamsters were therefore studied as a model for WEEV infection. Hamsters were highly sensitive to intraperitoneal (i.p.) infection with WEEV. Disease progression was rapid, and virus titers in serum, brain, liver, and kidney of infected hamsters peaked between 2 and 4 days post-virus inoculation (dpi). Foci of virus infection were detected in neurons of the cerebral cortex and midbrain. Pre-treatment i.p. with either interferon alfacon-1 (5 μg/kg/day) or with Ampligen® (3.2 mg/kg/day) resulted in complete survival, reduced brain titers, and improved weight gain. This model of WEEV infection in hamsters appears to serve as a suitable model for the evaluation of potential therapeutic agents for the treatment of WEE disease.

Treatment of West Nile Virus-Infected Mice with Reactive Immunoglobulin Reduces Fetal Titers and Increases Dam Survival

Justin G. Julander et al. — Thu, 15 Dec 2011 13:24:19 PST

The objectives of this study were to determine if injection of West Nile virus (WNV) into timed-pregnant mice would result in fetal infection and if administration of WNV-reactive immunoglobulin would increase dam survival and reduce fetal viral titers. Dams injected on 7.5 days post-coitus (dpc) had detectable viral titers in the placenta 10.5 dpc with a mean titer of 10^4.9 50% cell-culture infectious doses per gram of tissue (CCID50/g tissue). The mean placental titer increased to 10^8.6 CCID50/g tissue at 12.5 dpc. Infectious virus was detectable 12.5 dpc in 10 of 10 fetuses with a mean titer of 10^7.5 CCID50/g tissue. Treatment of dams (challenged with WNV on 7.5 dpc) with WNV-reactive human immunoglobulin (Ig) on 8.5 and 9.5 dpc resulted in a significant reduction of virus in fetuses as compared with non-reactive human Ig-treated females on 12.5 dpc (P ≤ 0.001). Treatment also resulted in survival of dams to term. Treatment of dams with WNV-reactive human Ig on 12.5 and 13.5 dpc also resulted in reduction of viral titer on 14.5 dpc, indicating that later treatment may also be efficacious. This suggests that Ig treatment may be useful in treating fetal WNV infection in women.

Characterization of Antiviral Activity of Entecavir in Transgenic Mice Expressing Hepatitis B Virus

Justin G. Julander et al. — Thu, 15 Dec 2011 13:24:18 PST

Entecavir (ETV), a cyclopentyl guanosine nucleoside analog, was evaluated in transgenic mice expressing hepatitis B virus (HBV). ETV administered orally once daily for 10 days at a dosage of 3.2 mg/kg significantly (P≤0.001) reduced liver HBV DNA in female mice from 5.9 to <0.82 pg of HBV DNA per μg of cellular DNA, and from 8.3 to <1.1 pg/μg in male mice. To compare the efficacy of ETV with other compounds previously evaluated in this model and with ETV activities in other animal models, the efficacy of serial one-half log dilutions of ETV were evaluated in both male and female mice to determine the minimal effective dose. End-point titration experiments resulted in a statistically significant HBV DNA reduction in the liver at concentrations of 0.032 and 0.1 mg/kg per day in female and male mice, respectively. Viral liver RNA, and serum e (HBeAg), serum surface (HBsAg), and liver core antigens (HBcAg) were not affected by ETV treatment presumably because the antiviral target was viral polymerase activity and the HBV produced from the transgene was not capable of secondary rounds of infection in the mouse. ETV was well tolerated and no morbidity or mortality was observed during the 10-day study. Similar to other animal models, ETV displayed potent anti-HBV activity in this transgenic mouse model.

Error-Prone Replication of West Nile Virus Caused by Ribavirin

C. W. Day et al. — Thu, 15 Dec 2011 13:24:16 PST

Ribavirin has been reported to cause error-prone replication and viral extinction in RNA viruses. The antiviral activity of ribavirin against West Nile virus (WNV) was evaluated in various cell lines to select a model in which mutagenic effects could be studied. The antiviral activity was greatest in HeLa cells as compared to CV-1, L929, Vero, or MA-104 cells. WNV was also passaged sequentially in cell monolayers treated with ribavirin to determine whether cumulative mutations could lead to viral extinction in these cell lines. The virus was abrogated in HeLa cells after 4 passages, while high viral titers persisted after many passages in other cells. A molecular clone of WNV was propagated in HeLa cells treated with 15 μg/mL ribavirin, and sequencing of viral genome segments revealed significant increases in transition mutations, demonstrating that ribavirin induced error-prone replication. The relative infectivity of viral RNA synthesized in the presence of ribavirin was shown to be reduced compared with untreated controls. These data support the hypothesis that error catastrophe is one of the modes of action for ribavirin against WNV.