Antiviral activity of the proteasome inhibitor VL-01 against influenza A viruses

The appearance of highly pathogenic avian influenza A viruses of the H5N1 subtype being able to infect humans and the 2009 H1N1 pandemic reveals the urgent need for new and efficient countermeasures against these viruses. The long-term efficacy of current antivirals is often limited, because of the emergence of drug-resistant virus mutants. A growing understanding of the virus-host interaction raises the possibility to explore alternative targets involved in the viral replication. In the present study we show that the proteasome inhibitor VL-01 leads to reduction of influenza virus replication in human lung adenocarcinoma epithelial cells (A549) as demonstrated with three different influenza virus strains, A/Puerto Rico/8/34 (H1N1) (EC₅₀ value of 1.7 μM), A/Regensburg/D6/09 (H1N1v) (EC₅₀ value of 2.4 μM) and A/Mallard/Bavaria/1/2006 (H5N1) (EC₅₀ value of 0.8 μM). In in vivo experiments we could demonstrate that VL-01-aerosol-treatment of BALB/c mice with 14.1 mg/kg results in no toxic side effects, reduced progeny virus titers in the lung (1.1 ± 0.3 log₁₀ pfu) and enhanced survival of mice after infection with a 5-fold MLD₅₀ of the human influenza A virus strain A/Puerto Rico/8/34 (H1N1) up to 50%. Furthermore, treatment of mice with VL-01 reduced the cytokine release of IL-α/β, IL-6, MIP-1β, RANTES and TNF-α induced by LPS or highly pathogen avian H5N1 influenza A virus. The present data demonstrates an antiviral effect of VL-01 in vitro and in vivo and the ability to reduce influenza virus induced cytokines and chemokines.     

Patchouli alcohol: in vitro direct anti-influenza virus sesquiterpene in <Emphasis Type="Italic">Pogostemon cablin</Emphasis> Benth.

During the screening of anti-influenza virus substances from traditional herbal medicines, the methanol extract from the leaves of Pogostemon cablin Benth. showed potent in vitro antiviral activity (99.8% inhibition at a concentration of 10 μg/mL) against influenza virus A/PR/8/34 (H1N1). The anti-influenza virus principle was isolated from the hexane-soluble fraction, through solvent fractionation, repeated silica gel column chromatography, and reversed-phase HPLC. The major active principle was a volatile substance that was identified as a sesquiterpene, patchouli alcohol (1), on the basis of its spectral analyses. When anti-influenza virus activity against A/PR/8/34 was evaluated by the plaque forming assay, patchouli alcohol reduced the number of plaques by 75% at 2 μg/mL and 89% at 10 μg/mL. Patchouli alcohol showed dose-dependent anti-influenza virus activity, and its IC₅₀ value was estimated to be 2.635 μM. Although 11 different sesquiterpenes were tested for antiviral activity against influenza virus A/PR/8/34, no or negligible activity was observed except for patchouli alcohol. Patchouli alcohol did not show anti-influenza virus activity against A/Guizhou/54/89 (H3N2), but showed weak activity against B/Ibaraki/2/85 (IC₅₀ = 40.82 μM). Patchouli alcohol did not show inhibitory activity against influenza virus neuraminidase.     

Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N

Cyanovirin-N (CV-N), a protein derived from Nostoc ellipsosporum, neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation sites. Recently we created a hybrid (reassortant) influenza A/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99 (H1N1) with an Asp225Gly mutation in the HA1, that was lethal to mice yet retained sensitivity to CV-N. We then utilized this model system to test the efficacy of CV-N against influenza. CV-N efficacy was dose-responsive from 0.0625 to 1 mg/kg/day when administered intranasally (i.n.) twice daily for 4 days starting 4 h prior to virus exposure. In a second study, survival benefit was seen with CV-N treatments (0.5 mg/kg/day for 4 days) beginning at −4 or +6 h, but was significantly reduced at +12 h. The early treatment resulted in up to 100% survival and 1000-fold reduction in lung virus titer on day 3 of the infection. In contrast, ribavirin (a positive control—75 mg/kg/day) treatment resulted in 30% survival and 30-fold decrease in lung virus titers. Lung consolidation scores and lung weights were significantly reduced by CV-N and ribavirin treatment on day 6 of the infection. Ferrets infected with a non-animal adapted influenza A/Charlottesville/31/95 (H1N1) virus were treated intranasally with CV-N (50 μg twice daily for 5 days starting 24 h before virus challenge). They exhibited 100-fold lower viral titers in nasal washes than placebos 1 day after treatment, but virus titers were equivalent on days 2–7. CV-N has the potential for prophylaxis and early initiation of treatment of influenza virus infections.     

Assessment of the efficacy of the neuraminidase inhibitor oseltamivir against 2009 pandemic H1N1 influenza virus in ferrets

Pandemic 2009 influenza A (H1N1) virus (H1N1pdm) is different from contemporary seasonal human viruses in that it can cause infection deep in the lungs of critical care patients. Here we establish a mammalian animal model and assessed the efficacy of the neuraminidase (NA) inhibitor oseltamivir treatment against H1N1pdm virus infection. Oseltamivir (25 mg/kg/day twice daily for 5 days) was orally administered to groups of ferrets, starting either 2 or 24 h after inoculation with 10⁶ PFU of A/California/04/2009 (H1N1) influenza virus. We determined that virus replication was restricted to 1 or 2 of 4 lung lobes in oseltamivir-treated animals, while virus was consistently isolated from 4 of 4 lung lobes in control animals (1.5-3.8 log₁₀ PFU/g). Analysis of arterial blood oxygenation revealed less pronounced changes in partial oxygen and carbon dioxide pressure in oseltamivir-treated ferrets, and histologic examination confirmed reduced pneumonia. Treated animals had significantly decreased inflammatory responses in the upper respiratory tract (P < 0.05), less fever and weight loss, and less reduction of activity. Virus titers in the nasal washes of treated and control ferrets did not differ significantly. NA sequencing and fluorescence-based phenotypic assays identified no oseltamivir-resistant variants. Overall, oseltamivir treatment decreases the signs of infection and reduced the spread of H1N1pdm influenza virus in the lungs of ferrets and therefore impeded the development of viral pneumonia.     

Protective effect of T3 peptide,an active fragment of tumstatin,against ischemia/reperfusion injury in rat heart

Ischemia/reperfusion (I/R)-induced oxidative stress is a serious clinical problem in the reperfusion therapy for ischemic diseases. Tumstatin is an endogenous bioactive peptide cleaved from type IV collagen α3 chain. We previously reported that T3 peptide, an active subfragment of tumstatin, exerts cytoprotective effects on H₂O₂-induced apoptosis through the inhibition of intracellular reactive oxygen species (ROS) production in H9c2 cardiomyoblasts. In this study, we investigated whether T3 peptide has cardioprotective effects against I/R injury by using in vitro and ex vivo experimental models. H9c2 cardiomyoblasts were stimulated with oxygen and glucose deprivation (OGD) for 12 h followed by reoxygenation for 1–8 h (OGD/R; in vitro model). The cells were treated with T3 peptide (30–1000 ng/ml) during OGD. Ten minutes after the pre-perfusion of T3 peptide (300 ng/ml), Langendorff perfused rat hearts were exposed to ischemia for 30 min followed by reperfusion for 1 h (ex vivo model). T3 peptide inhibited OGD/R-induced apoptosis through the inhibition of mitochondrial ROS production and dysfunction in H9c2 cardiomyoblasts. T3 peptide also prevented I/R-induced cardiac dysfunction, arrhythmia and myocardial infarction in the perfused rat heart. In conclusion, we for the first time demonstrated that T3 peptide exerts cardioprotective effects against I/R injury.     

新型甲型H1N1流感病毒神经氨酸酶抑制剂细胞水平评价体系的建立

本文旨在建立以2009新型甲型H1N1流感病毒神经氨酸酶 (neuraminidase, NA) 为靶点的神经氨酸酶抑制剂 (neuraminidase inhibitors, NAIs) 细胞水平评价体系。NA有促进流感病毒释放的作用, 本研究应用重组病毒技术, 通过将表达NA [A/California/04/2009 (H1N1)] 的质粒、表达流感病毒血凝素蛋白HA (hemagglutinin) 的质粒以及表达敲除外壳基因并带有荧光素酶报告基因的HIV-1基因组共转染至病毒生成细胞, 产生以HA、NA为外壳蛋白包裹HIV-1核心的重组病毒。在病毒释放前加入不同浓度的化合物, 收集病毒上清液感染细胞, 通过测定感染率来反映化合物对重组病毒NA的抑制作用。经用阳性对照药物奥司他韦及其羧酸盐证实本模型能够反映化合物对病毒NA的抑制作用; 在此基础上, 本研究还建立了奥司他韦耐药株评价模型。本研究所建立的体系可用于针对新型甲型H1N1流感病毒及其临床耐药株神经氨酸酶抑制剂的寻找和评价。     

In vitro metabolism of the new antifungal dapaconazole using liver microsomes

Dapaconazole is a new antifungal imidazole that has been shown a high efficacy against several pathogenic fungi. This study aimed to investigate the interspecies variation in the in vitro metabolic profiles and in vivo hepatic clearance (CL_H,in vivo) prediction of dapaconazole using liver microsomes from male Sprague Dawley rat, male Beagle dog and mixed gender human using a liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method. In addition, the produced metabolites were identified by ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS/MS). The microsomal protein concentration of 0.1 mg/mL and the incubation time of 10 min were employed for the kinetics determination, resulting in a sigmoidal kinetic profile for all species evaluated. The predicted CL_H,in vivo was 6.5, 11.6 and 7.5 mL/min/kg for human, rat and dog, respectively. Furthermore, five metabolized products were identified. These findings provide preliminary information for understanding dapaconazole metabolism and the interspecies differences in catalytic behaviours, supporting the choice of a suitable laboratory animal for future pharmacokinetics and metabolism studies.     

Inactivation of high and low pathogenic avian influenza virus H5 subtypes by copper ions incorporated in zeolite-textile materials

The effect of cotton textiles containing Cu²⁺ held by zeolites (CuZeo-textile) on the inactivation of H5 subtype viruses was examined. Allantoic fluid (AF) containing a virus (AF virus) (0.1 ml) was applied to the textile (3 × 3-cm), and incubated for a specific period at ambient temperature. After each incubation, 0.9 ml of culture medium was added followed by squeezing to recover the virus into the medium. The recovered virus was titrated using Madin-Darby canine kidney (MDCK) cells or 10-day-old embryonated chicken eggs.The highly pathogenic H5N1 and the low pathogenic H5N3 viruses were inactivated on the CuZeo-textile, even after short incubation. The titer of A/chicken/Yamaguchi/7/04 (H5N1) in MDCK cells and in eggs declined by >5.0 log₁₀ and 5.0 log₁₀, respectively, in 30 s. The titer of A/whooper swan/Hokkaido/1/08 (H5N1) in MDCK cells declined by 2.3 and 3.5 in 1 and 5 min, respectively. When A/whistling swan/Shimane/499/83 (H5N3) was treated on the CuZeo-textile for 10 min, the titer declined by >5.0 log₁₀ in MDCK cells and by >3.5 log₁₀ in eggs. In contrast, no decrease in the titers was observed on cotton textiles containing zeolites alone (Zeo-textile). Neither cytopathic effects nor NP antigens were detected in MDCK cells inoculated with the H5N1 virus treated on the CuZeo-textile. The viral genes (H5, N1, M, and NP) were amplified from the virus treated on the CuZeo-textile by RT-PCR. The hemagglutinating activity of the CuZeo-textile treated virus was unaffected, indicating that virus-receptor interactions were maintained. Electron microscopic analysis revealed a small number of particles with morphological abnormalities in the H5N3 virus samples recovered immediately from the CuZeo-textile, while no particles were detectable in the 10-min treated sample, suggesting the rapid destruction of virions by the Cu²⁺ in the CuZeo-textile. The loss of infectivity of H5 viruses could, therefore, be due to the destruction of virions by Cu²⁺. Interestingly, CuCl₂ treatment (500 and 5000 μM) did not have an antiviral effect on the AF viruses (H5N1 and H5N3) even after 48 h of incubation, although the titer of the purified H5N3 virus treated with CuCl₂ declined greatly. The antiviral effect was inhibited by adding the AF to the purified H5N3 virus prior to the CuCl₂ treatment. The known antibacterial/antifungal activities of copper suggest that the CuZeo-textile can be applied at a high level of hygiene in both animals and humans.     

Dynamics of antiviral-resistant influenza viruses in the Netherlands, 2005–2008

In the Netherlands, influenza specific antivirals are used for the therapy of influenza in nursing homes and hospitals, for prophylaxis in high risk groups and neuraminidase inhibitors are stockpiled as part of pandemic preparedness plans. To monitor the antiviral susceptibility profile, human influenza virus isolates derived from the Dutch influenza surveillance in 2005–2006 (n = 87), 2006–2007 (n = 58) and 2007–2008 (n = 128) were analyzed with phenotypic assays and sequencing. For adamantanes, a high proportion (>74%) of A(H3N2) viruses had the S31N mutation in M2 protein, while variation in the HA₁ region of adamantane-sensitive viruses suggested that adamantane-sensitive variants were reseeded into the Dutch population and re-emerged as drug-sensitive due to M-segment reassortment. For neuraminidase inhibitors oseltamivir and zanamivir, 98% of types A and B influenza viruses prior to 2007–2008 were sensitive for both, whereas 24% of the A(H1N1) viruses obtained in 2007–2008 were oseltamivir-resistant while retaining sensitivity to zanamivir and adamantanes. Furthermore, oseltamivir-resistant A(H1N1) or adamantane-resistant A(H3N2) virus infections were not associated with differences in clinical symptoms compared to infections with sensitive variants. Our data show the dynamic nature of emergence of drug-resistant influenza viruses, stressing the need for surveillance of resistance trends as part of influenza monitoring programs.     

某区甲型H1N1流感病毒分离鉴定及同源性的分析

目的检测并分离甲型H1N1流感病毒,对开封地区首次分离到的病毒株进行全基因组序列测定及同源性分析,为研究流感病毒的流行及变异规律提供科学依据。方法采用Real-time RT-PCR方法检测,筛选确定出甲型H1N1流感病毒阳性标本;利用狗肾传代细胞分离得到甲型H1N1流感病毒株A/Kaifeng/01/2009(H1N1);测定并分析其全基因组序列;利用序列比对进行了同源性分析。结果从1828份流感样病例中检出甲型H1N1流感病毒阳性标本286份,阳性率15.6%。在开封地区首次获得甲型H1N1流感病毒株及全基因组序列。基因组序列分析表明:该毒株与2009年大流行株高度同源,为同一进化分支。与以往流行的猪流感病毒株对比发现,HA基因有12个碱基发生了点突变。结论 MDCK细胞对甲型H1N1流感病毒具有较高敏感性;开封地区首例甲型H1N1流感病例分离病毒株与北美流行株高度同源;相对于以往古典型猪流感代表株出现了HA蛋白抗原性漂移;为今后进一步开展甲型H1N1流感病毒分子生物学研究奠定基础。     

Mouse compound muscle action potential assay: An alternative method to conduct the LD50 botulinum toxin type A potency test

Purpose

We performed a prevalidation of the compound muscle action potential (CMAP) assay to determine the potency of botulinum neurotoxin type A (BoNT/A) with the aim of substituting for the mouse lethality test (LD₅₀), which is used for quality control.

Methods

Prevalidation experiments were performed to demonstrate the specificity, linearity, accuracy, precision, range, limit of quantitation (LOQ), and robustness of the assay. For specificity, toxin detection ability was determined in the presence of neutralizing antibodies (0.8 and 8 IU/mL). Linearity of this assay was determined by measuring CMAP amplitude using nine concentrations (n = 3) in the range of 1-100 U/mL (n = 3). Accuracy was assessed using five concentrations (n = 3) in the range of 4-40 U/mL. Intermediate precision was confirmed by analyzing individually prepared reagents on multiple days by one operator (n = 3). Different body weights (23-25 and 25-27 g) and measurement times (3-5 and 5-7 min) after anesthetic induction were tested to assess robustness.

Results

This assay might have BoNT/A specificity, based on the CMAP amplitude recovery using a concentration of neutralizing antibodies. The calibration curves were linear over the range of 2-40 U/mL (R² = 0.982). The accuracy of 14 determinations was within the range of 89.8-118.6% compared to the theoretical values among 15 determinations, except one (131.3%). Assay variability was acceptable with coefficients of variation of 4.3-14.4%. The range of quantification and the LOQ were 4-40 U/mL and 4 U/mL, respectively. Different body weights and measurement times after inducing anesthesia had no effect on CMAP amplitude.

Conclusions

These results suggest that the mouse CMAP assay is an alternative method to the standard LD₅₀ potency test and meets the requirement of the three Rs (particularly refinement and reduction).     

Elevated plasma Pim‐1 and its clinical significance in patients with pulmonary arterial hypertension

This study was aimed to determine plasma Pim‐1 levels in patients with pulmonary arterial hypertension (PAH) and to estimate the clinical value of Pim‐1 as a biomarker of PAH. This was a single‐centre retrospective study in 111 patients with congenital heart disease (CHD) and idiopathic PAH (IPAH). Those CHD patients were divided into two groups: PAH associated with CHD (PAH‐CHD) and CHD without PAH (nPAH‐CHD). Plasma Pim‐1 levels were measured by enzyme‐linked immunosorbent assay. (a) Plasma Pim‐1 levels were significantly increased in patients with PAH‐CHD and IPAH compared with the healthy control group (27.81 ± 11.34 ng/mL vs 13.02 ± 5.30 ng/mL; 32.81 ± 12.28 ng/mL vs 13.02 ± 5.30 ng/mL, P < 0.05) and nPAH‐CHD (27.81 ± 11.34 ng/mL vs 17.33 ± 7.99 ng/mL; 32.81 ± 12.28 ng/mL vs 17.33 ± 7.99 ng/mL, P < 0.05). Pim‐1 levels were substantially increased in patients with severe PAH‐CHD compared with mild‐to‐moderate PAH‐CHD (19.12 ± 6.70 ng/mL vs 8.54 ± 3.71 ng/mL, P < 0.05). (b) Pim‐1 levels were correlated positively with the mean pulmonary artery pressure (mPAP) and pulmonary vascular resistance (PVR) (r = 0.582, 0.516; P < 0.001, respectively), while negatively with tricuspid annular plane systolic excursion (TAPSE), tricuspid annular plane systolic velocity (S’) and right ventricular fractional area changes (RVFAC) (r = ?0.375, ?0.354, ?0.507; P < 0.05, respectively). (c) PAH‐CHD and severe PAH‐CHD was identified by plasma Pim‐1 with a cutoff value of 16.8 ng/mL (P < 0.001) with a sensitivity of 87.3% and a specificity of 65%, and a cutoff value of 20.53 ng/mL (P < 0.001) with a sensitivity of 87.3% and a specificity of 52%, respectively. Plasma Pim‐1 levels were significantly higher in patients with PAH‐CHD and IPAH. Plasma Pim‐1 may represent an effectively biomarker in patients with PAH.     

Immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing HA of H9N2 avain influenza virus and chicken IL-18

Control of the circulation of H9N2 avian influenza virus (AIV) is a major concern for both animal and public health, and H9N2 AIV poses a major threat to the chicken industry worldwide. Here, we developed a recombinant fowlpox virus (rFPV-HA) expressing the haemagglutinin (HA) gene of the A/CH/JY/1/05 (H9N2) influenza virus and a recombinant fowlpox virus (rFPV-HA/IL18) expressing the HA gene and chicken interleukin-18 (IL-18) gene. Recombinant plasmid pSY-HA/IL18 was constructed by cloning chicken IL-18 expression cassette into recombinant plasmid pSY-HA containing the HA gene. Two rFPVs were generated by transfecting two recombinant plasmids into the chicken embryo fibroblast cells pre-infected with S-FPV-017, and assessed for their immunological efficacy on one-day-old White Leghorn specific-pathogen-free chickens challenged with the A/CH/JY/1/05 (H9N2) strain. There was a significant difference in HI antibody levels (P < 0.05) elicited by either rFPV-HA or rFPV-HA/IL18. The level of splenocyte proliferation response in the rFPV-HA/IL18-vaccinated group was significantly higher (P < 0.05) than that in the rFPV-HA group. After challenge with 10^6.5 ELD₅₀ H9N2 AIV 43 days after immunization, rFPVs vaccinated groups could prevent virus shedding and replication in multiple organs in response to H9N2 AIV infection, and rFPV-HA/IL18 vaccinated group had better inhibition of viruses than rFPV-HA vaccinated group. Our results show that the protective efficacy of the rFPV-HA vaccine could be enhanced significantly by simultaneous expression of IL-18.     

Synthesis and Biological Evaluation of 2-oxo-pyrazine-3-carboxamide-yl Nucleoside Analogues and Their Epimers as Inhibitors of Influenza A Viruses

Novel 2-oxo-pyrazine-3-carboxamide-yl nucleoside analogues and their epimers were designed, synthesized and evaluated for their activities against influenza A viruses H1N1 and H3N2 in Madin-Darby canine kidney cells. All the compounds showed low cytotoxicities in these anti-influenza tests. One of the epimers, 4-[(1S, 3R, 4R, 7R)-7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-3-oxo-3,4-dihydropyrazine-2-carboxamide 8 a, with high antiviral activities (IC₅₀ = 7.41, 5.63 μm for H3N2 and H1N1, respectively) and remarkable low cytotoxicity (TC₅₀ > 200 μm ), has great potential for further development as a novel anti-influenza A agent. Molecular docking of compound 8a with RNA-dependent RNA polymerase was performed to understand the binding mode between these inhibitors and the active site of RdRp and to rationalize some SARs.     

In vitro and in vivo efficacy of fluorodeoxycytidine analogs against highly pathogenic avian influenza H5N1, seasonal, and pandemic H1N1 virus infections

Various fluorodeoxyribonucleosides were evaluated for their antiviral activities against influenza virus infections in vitro and in vivo. Among the most potent inhibitors was 2′-deoxy-2′-fluorocytidine (2′-FdC). It inhibited various strains of low and highly pathogenic avian influenza H5N1 viruses, pandemic H1N1 viruses, an oseltamivir-resistant pandemic H1N1 virus, and seasonal influenza viruses (H3N2, H1N1, influenza B) in MDCK cells, with the 90% inhibitory concentrations ranging from 0.13 to 4.6 μM, as determined by a virus yield reduction assay. 2′-FdC was then tested for efficacy in BALB/c mice infected with a lethal dose of highly pathogenic influenza A/Vietnam/1203/2004 H5N1 virus. 2′FdC (60 mg/kg/d) administered intraperitoneally (i.p.) twice a day beginning 24 h after virus exposure significantly promoted survival (80% survival) of infected mice (p = 0.0001). Equally efficacious were the treatment regimens in which mice were treated with 2′-FdC at 30 or 60 mg/kg/day (bid X 8) beginning 24 h before virus exposure. At these doses, 70-80% of the mice were protected from death due to virus infection (p = 0.0005, p = 0.0001; respectively). The lungs harvested from treated mice at day four of the infection displayed little surface pathology or histopathology, lung weights were lower, and the 60 mg/kg dose reduced lung virus titers, although not significantly compared to the placebo controls. All doses were well tolerated in uninfected mice. 2′-FdC could also be administered as late as 72 h post virus exposure and still significantly protect 60% mice from the lethal effects of the H5N1 virus infection (p = 0.019). Other fluorodeoxyribonucleosides tested in the H5N1 mouse model, 2′-deoxy-5-fluorocytidine and 2′-deoxy-2′,2′-difluorocytidine, were very toxic at higher doses and not inhibitory at lower doses. Finally, 2′-FdC, which was active in the H5N1 mouse model, was also active in a pandemic H1N1 influenza A infection model in mice. When given at 30 mg/kg/d (bid X 5) beginning 24 h before virus exposure), 2′-FdC also significantly enhanced survival of H1N1-infected mice (50%, p = 0.038) similar to the results obtained in the H5N1 infection model using a similar dosing regimen (50%, p < 0.05). Given the demonstrated in vitro and in vivo inhibition of avian influenza virus replication, 2′FdC may qualify as a lead compound for the development of agents treating influenza virus infections.     

Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N

Cyanovirin-N (CV-N), a protein derived from Nostoc ellipsosporum, neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation sites. Recently we created a hybrid (reassortant) influenza A/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99 (H1N1) with an Asp225Gly mutation in the HA1, that was lethal to mice yet retained sensitivity to CV-N. We then utilized this model system to test the efficacy of CV-N against influenza. CV-N efficacy was dose-responsive from 0.0625 to 1 mg/kg/day when administered intranasally (i.n.) twice daily for 4 days starting 4 h prior to virus exposure. In a second study, survival benefit was seen with CV-N treatments (0.5 mg/kg/day for 4 days) beginning at −4 or +6 h, but was significantly reduced at +12 h. The early treatment resulted in up to 100% survival and 1000-fold reduction in lung virus titer on day 3 of the infection. In contrast, ribavirin (a positive control—75 mg/kg/day) treatment resulted in 30% survival and 30-fold decrease in lung virus titers. Lung consolidation scores and lung weights were significantly reduced by CV-N and ribavirin treatment on day 6 of the infection. Ferrets infected with a non-animal adapted influenza A/Charlottesville/31/95 (H1N1) virus were treated intranasally with CV-N (50 μg twice daily for 5 days starting 24 h before virus challenge). They exhibited 100-fold lower viral titers in nasal washes than placebos 1 day after treatment, but virus titers were equivalent on days 2–7. CV-N has the potential for prophylaxis and early initiation of treatment of influenza virus infections.     

2‐Pyridinyl‐4(3H)‐Quinazolinone: A Scaffold for Anti‐influenza A Virus Compounds

A series of 2‐pyridinyl‐3‐substituted‐4(3H)‐quinazolinones were synthesized, and their anti‐influenza A virus activities were determined using the cytopathic effect inhibition assay. Most of the compounds were potent with IC₅₀ values ranging from 51.6 to 93.0 μm , which are better than that of the currently marketed drug ribavirin. The molecular mechanisms of the new compounds were investigated using neuraminidase inhibition assay, cellular NF‐κB signaling pathway inhibition assay, and computational docking. Compound 4e , which is a N3 imidazol‐1‐ylpropyl‐substituted derivative of 2‐pyridinyl‐4(3H)‐quinazolinone, had the most potent anti‐influenza A virus activity in vitro, and inhibited both virus neuraminidase and cellular NF‐κB signaling pathway. In conclusion, 2‐pyridinyl‐4(3H)‐quinazolinone is a new scaffold for the design of potent anti‐influenza A virus compounds, offering an alternative approach to tackle influenza drug resistance.     

Actions of the Kunitz-type serine protease inhibitor Amblyomin-X on VEGF-A-induced angiogenesis

Amblyomin-X is a Kunitz-type serine protease inhibitor (Kunitz-type SPI) designed from the cDNA library of the Amblyomma cajennense tick, which displays in vivo anti-tumor activities. Here, the mechanisms of actions of Amblyomin-X in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis were characterized. Topical application of Amblyomin-X (10 or 100 ng/10 μl; each 48 h) inhibited VEGF-A-induced (10 ng/10 μl; each 48 h) angiogenesis in the dorsal subcutaneous tissue in male Swiss mice. Moreover, similar effect was observed in the VEGF-A-induced angiogenesis in the chicken chorioallantoic membrane (CAM). Additional in vitro assays in t-End cells showed that Amblyomin-X treatment delayed the cell cycle, by maintaining them in G0/G1 phase, and inhibited cell proliferation and adhesion, tube formation and membrane expression of the adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1), regardless of mRNA synthesis. Together, results herein reveal the role of Kunitz-type SPI on in vivo VEGF-A-induced angiogenesis, by exerting modulatory actions on endothelial cell proliferation and adhesion, especially on membrane expression of PECAM-1. These data provide further mechanisms of actions of Kunitz-type SPI, corroborating their relevance as scientific tools in the design of therapeutic molecules.     

Bactericidal activity of daptomycin versus vancomycin in the presence of human albumin against vancomycin-susceptible but tolerant methicillin-resistant Staphylococcus aureus (MRSA) with daptomycin minimum inhibitory concentrations of 1–2 μg/mL

This study explored the influence of vancomycin tolerance and protein binding on the bactericidal activity of vancomycin versus daptomycin (protein binding 36.9% vs. 91.7%, respectively) against four vancomycin-tolerant methicillin-resistant Staphylococcus aureus (MRSA) [minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) = 0.5/16, 1/32, 2/32 and 1/32 μg/mL for vancomycin and 1/1, 1/2, 2/2 and 2/4 μg/mL for daptomycin]. Killing curves were performed with vancomycin/daptomycin concentrations equal to serum peak concentrations (C_max) (65.70/98.60 μg/mL) and trough concentrations (C_min) (7.90/9.13 μg/mL) in the presence and absence of a physiological human albumin concentration (4 g/dL), controlled with curves with the theoretical free drug fraction of vancomycin/daptomycin C_max (41.45/8.18 μg/mL) and C_min (4.98/0.76 μg/mL). Vancomycin C_max and C_min concentrations, regardless of the media, showed a bacteriostatic profile not reaching a reduction of 99% or 99.9% of the initial inocula during the 24-h experimental time period. Daptomycin antibacterial profiles significantly differed when testing C_max and C_min. C_max was rapidly bactericidal (≤4 h) with >5 log₁₀ reduction in the initial inocula for all strains, regardless of the presence or not of albumin or the use of concentrations similar to free C_max. C_min exhibited similar final colony counts at 0 h and 24 h in curves with albumin, but with >3 log colony-forming units (CFU)/mL reduction at ≤4 h for strains with an MIC of 1 μg/mL and ca. 2 log CFU/mL reduction at ≤6 h for strains with an MIC of 2 μg/mL. This activity was significantly higher than the activity of the free C_min fraction. The results of this study reinforce the idea that pharmacodynamics using concentrations calculated using reported protein binding are unreliable. Daptomycin exhibited rapid antibacterial activity against vancomycin-tolerant MRSA isolates even against those with high daptomycin MICs in the presence of physiological albumin concentrations.