Antiviral activity of Epimedium koreanum Nakai water extract against influenza viruses.

Epimedium koreanum Nakai (EKN) is a popular plant in Korean and Chinese medicine for treating a variety of ailments. The aqueous extract of EKN has a significant inhibitory impact on influenza A virus (IAV) infection by directly blocking viral attachment and having a virucidal effect, according to this study. Using fluorescent microscopy and fluorescence-activated cell sorting (FACS) with a green fluorescent protein (GFP)-tagged Influenza A/PR/8/34 virus, we examined the effect of EKN on viral infection. By viral infection, EKN strongly suppresses GFP expression, and at a dosage of 100 µg/mL, EKN decreased GFP expression by up to 90% of the untreated infected control. Immunofluorescence and Western blot analyses against influenza viral proteins revealed that EKN decreased influenza viral protein expression in a dose-dependent manner. EKN inhibited the H1N1 influenza virus's hemagglutinin (HA) and neuraminidase (NA), preventing viral attachment to cells. Furthermore, EKN had a virucidal impact and inhibited the cytopathic effects of H1N1, H3N2 and influenza B virus infection. Finally, our findings show that EKN has the potential to be developed as a natural viral inhibitor against influenza virus infection.

Neuraminidase 1 and its Inhibitors from Chinese Herbal Medicines: An Emerging Role for Cardiovascular Diseases.

Neuraminidase, also known as sialidase, is ubiquitous in animals and microorganisms. It is predominantly distributed in the cell membrane, cytoplasmic vesicles, and lysosomes. Neuraminidase generally recognizes the sialic acid glycosidic bonds at the ends of glycoproteins or glycolipids and enzymatically removes sialic acid. There are four types of neuraminidases, named as Neu1, Neu2, Neu3, and Neu4. Among them, Neu1 is the most abundant in mammals. Recent studies have revealed the involvement of Neu1 in several diseases, including cardiovascular diseases, diabetes, cancers, and neurological disorders. In this review, we center the attention to the role of Neu1 in cardiovascular diseases, including atherosclerosis, ischemic myocardial injury, cerebrovascular disease, congenital heart disease, and pulmonary embolism. We also summarize inhibitors from Chinese herbal medicines (CHMs) in inhibiting virus neuraminidase or human Neu1. Many Chinese herbs and Chinese herb preparations, such as Lonicerae Japonicae Flos, Scutellariae Radix, Yupingfeng San, and Huanglian Jiedu Decoction, have neuraminidase inhibitory activity. We hope to highlight the emerging role of Neu1 in humans and potentially titillate interest for further studies in this area.

Synthesis of novel derivatives of 7,8-dihydro-6H-imidazo[2,1-b][1,3]benzothiazol-5-one and their virus-inhibiting activity against influenza A virus.

Influenza remains a highly pathogenic and hardly controlled human infection. The ability of selecting drug-resistant variants necessitates the search and development of novel anti-influenza drugs. Herein, we describe the synthesis and evaluation of a series of novel 2-substituted 7,8-dihydro-6H-imidazo[2,1-b][1,3]benzothiazol-5-ones 3a-k for their virus-inhibiting activity against influenza A virus. The new analogues 3a-k prepared in two steps from commercially available cyclohexane-1,3-diones were fully characterized by their NMR and mass spectral data. Among the new derivatives screened for cytotoxicity and in vitro antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells, three analogues 3i-k containing a thiophene unit were found to exhibit high virus-inhibiting activity (high SI values) and a favorable toxicity profile. The compound 3j (CC50 : >1000 µM, SI = 77) with higher potency is the best anti-influenza hit analogue for further structural optimization and drug development. The most active compounds did not inhibit viral neuraminidase and possess therefore other targets and mechanisms of activity than the currently used neuraminidase inhibitors.

Sheng Jiang San, a traditional multi-herb formulation, exerts anti-influenza effects in vitro and in vivo via neuraminidase inhibition and immune regulation.

BACKGROUND: Sheng Jiang San (SJS), a multi-herb formulation, is used in treating high fever, thirsty and anxiety in ancient China and it is sometimes used to treat seasonal influenza nowadays. However, there is no evidence-based investigation and mechanism research to support the anti-influenza efficacy of SJS. This study aims at evaluating the anti-influenza effect of SJS and investigating its possible mechanism. METHODS: The inhibitory effect of SJS against different influenza virus strains on MDCK cells was examined. Influenza virus infected BALB/c mice were employed to evaluate the efficacy as in vivo model. Mice challenged with A/PR/8/34 (H1N1) were orally administrated 1 g/kg/day of SJS for seven days and monitored for 14 days. The survival rate, body weight changes, lung index, lung viral load, histopathologic changes and immune regulation of the mice were measured. The underlying anti-influenza virus mechanism of SJS was studied by a series of biological assays to determine if hemagglutinin, ribonucleoprotein complex or neuraminidase were targets of SJS. RESULTS: Results showed SJS exerted a broad-spectrum of inhibitory effects on multiple influenza strains in a dose-dependent manner. IC50 of SJS against A/WSN/33 (H1N1) was lower than 35 µg/ml. SJS also protected 50% of mice from A/PR/8/34 (H1N1) infection. The lung index and the lung viral load of SJS treated mice were significantly decreased compared with untreated mice. Meanwhile, SJS targeted on neuraminidase of influenza virus as SJS at 2 mg/ml inhibited 80% of neuraminidase enzymatic activity. SJS also significantly down-regulated TNF-α and up-regulated IL-2 of influenza virus induced mice. CONCLUSIONS: Thus, SJS is a useful formulation for treating influenza virus infection.

A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets.

Low pathogenic H7N9 influenza viruses have recently evolved to become highly pathogenic, raising concerns of a pandemic, particularly if these viruses acquire efficient human-to-human transmissibility. We compared a low pathogenic H7N9 virus with a highly pathogenic isolate, and two of its variants that represent neuraminidase inhibitor-sensitive and -resistant subpopulations detected within the isolate. The highly pathogenic H7N9 viruses replicated efficiently in mice, ferrets, and/or nonhuman primates, and were more pathogenic in mice and ferrets than the low pathogenic H7N9 virus, with the exception of the neuraminidase inhibitor-resistant virus, which showed mild-to-moderate attenuation. All viruses transmitted among ferrets via respiratory droplets, and the neuraminidase-sensitive variant killed several of the infected and exposed animals. Neuraminidase inhibitors showed limited effectiveness against these viruses in vivo, but the viruses were susceptible to a polymerase inhibitor. These results suggest that the highly pathogenic H7N9 virus has pandemic potential and should be closely monitored.

Effects of corticosteroid and neuraminidase inhibitors on survival in patients with respiratory distress induced by influenza virus.

BACKGROUND/PURPOSE: Neuraminidase inhibitors (NAIs) including oseltamivir and peramivir are used for influenza treatment. A systemic corticosteroid is usually administrated for acute respiratory distress syndrome. The aim of this study was to investigate the effect of a systemic corticosteroid and its interaction with NAIs in patients with influenza infection and respiratory distress. METHODS: A retrospective survey of hospitalized patients infected with influenza from January 2012 to May 2014 was conducted in a medical center in Taiwan. RESULTS: Eighty-six patients were hospitalized during the study period. Forty-eight patients had respiratory distress and 39 of them (81.3%, 39/48) were supported by a mechanical ventilator. All patients with respiratory distress received oseltamivir; 60.4% (29/48) and 31.3% (15/48) of them received a corticosteroid and salvage intravenous peramivir, respectively. All-cause mortality was 29.1% (14/48), 20% (3/15), and 31% (9/29) in patients with respiratory distress, patients who received salvage peramivir, and patients who received a systemic corticosteroid, respectively. Salvage peramivir seemed to improve prognosis in patients with H1pdm09 or type B virus infection and respiratory distress (p = 0.05). Early initiating corticosteroid had a worse prognosis than initiation after 72 hours of NAI treatment (p = 0.024). In particular, a systemic corticosteroid seemed to lead to a shorter survival time in patients with chronic lung disease (p = 0.05). CONCLUSION: Salvage peramivir provided a better prognosis than monotherapy with oseltamivir in patients who were infected with H1pdm09 or type B virus and who developed respiratory distress. A systemic corticosteroid should be administered after initiating NAI therapy, especially in patients with chronic lung disease.

Oseltamivir PK/PD Modeling and Simulation to Evaluate Treatment Strategies against Influenza-Pneumococcus Coinfection.

Influenza pandemics and seasonal outbreaks have shown the potential of Influenza A virus (IAV) to enhance susceptibility to a secondary infection with the bacterial pathogen Streptococcus pneumoniae (Sp). The high morbidity and mortality rate revealed the poor efficacy of antiviral drugs and vaccines to fight IAV infections. Currently, the most effective treatment for IAV is by antiviral neuraminidase inhibitors. Among them, the most frequently stockpiled is Oseltamivir which reduces viral release and transmission. However, effectiveness of Oseltamivir is compromised by the emergence of resistant IAV strains and secondary bacterial infections. To date, little attention has been given to evaluate how Oseltamivir treatment strategies alter Influenza viral infection in presence of Sp coinfection and a resistant IAV strain emergence. In this paper we investigate the efficacy of current approved Oseltamivir treatment regimens using a computational approach. Our numerical results suggest that the curative regimen (75 mg) may yield 47% of antiviral efficacy and 9% of antibacterial efficacy. An increment in dose to 150 mg (pandemic regimen) may increase the antiviral efficacy to 49% and the antibacterial efficacy to 16%. The choice to decrease the intake frequency to once per day is not recommended due to a significant reduction in both antiviral and antibacterial efficacy. We also observe that the treatment duration of 10 days may not provide a clear improvement on the antiviral and antibacterial efficacy compared to 5 days. All together, our in silico study reveals the success and pitfalls of Oseltamivir treatment strategies within IAV-Sp coinfection and calls for testing the validity in clinical trials.

Treatment and prophylaxis of influenza and the problem of resistance to neuraminidase inhibitors.

Influenza virus neuraminidase inhibitors (NAIs), including oseltamivir, zanamivir and peramivir, are important antivirals for the treatment and prophylaxis of influenza. Increasing use of NAIs brings into focus the risk of drug resistance. The problem of resistance is of high clinical and epidemiological importance. There are generally three levels of antiviral resistance according to the way that resistance can be detected or inferred: genotypic, phenotypic and clinical resistance. Recently the problem of resistance to NAIs, although still rare (<2% of influenza isolates), has been rising. It should be underlined that NAI resistance in influenza viruses is relative, and despite its presence patients with resistant viruses may still benefit from receiving NAIs. The clinical resistance and the response to treatment with antivirals remain the most important proof of antiviral effectiveness. Currently, there has not been observed cross-resistance between oseltamivir and zanamivir, which may be a consequence of the number of given doses, differences in drug structure and duration of the drug concentrations in the site of infection. Early treatment with appropriate doses of NAI is necessary to minimize the likelihood of a resistant virus arising.

Potential therapeutic use of herbal extracts in trypanosomiasis.

The aim of the present study was to evaluate the effects of crude extracts from Handroanthus impetiginosa, Ageratum conyzoides, and Ruta graveolens on Leishmania amazonensis and Trypanosoma cruzi infection in vitro. The results showed that the extracts caused significant toxicity in promastigotes and trypomastigotes. A significant decrease in the rate of cell invasion by pretreated trypomastigotes and promastigotes was also observed. The extracts caused a significant reduction of the multiplication of intracellular amastigotes of both parasites. Therefore, these herbal extracts may be potential candidates for the development of drugs for the treatment of leishmaniasis and Chagas disease.

Computer-guided approach to access the anti-influenza activity of licorice constituents.

Neuraminidase (NA), a key enzyme in viral replication, is the first-line drug target to combat influenza. On the basis of a shape-focused virtual screening, the roots of Glycyrrhiza glabra (licorice) were identified as plant species with an accumulation of constituents that show 3D similarities to known influenza NA inhibitors (NAIs). Phytochemical investigation revealed 12 constituents identified as (E)-1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-3-(8-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)-2-propen-1-one (1), 3,4-dihydro-8,8-dimethyl-2H,8H-benzo[1,2-b:3,4-b']dipyran-3-ol (2), biochanin B (3), glabrol (4), glabrone (5), hispaglabridin B (6), licoflavone B (7), licorice glycoside B (8), licorice glycoside E (9), liquiritigenin (10), liquiritin (11), and prunin (12). Eleven of these constituents showed significant influenza virus NA inhibition in a chemiluminescence (CL)-based assay. Additional tests, including (i) a cell-based cytopathic effect inhibition assay (general antiviral activity), (ii) the evaluation of cytotoxicity, (iii) the inhibition of the NA of Clostridium perfringens (CL- and fluorescence (FL)-based assay), and (iv) the determination of self-fluorescence and quenching, provided further perspective on their anti-influenza virus potential, revealing possible assay interference problems and false-positive results. Compounds 1, 3, 5, and 6 showed antiviral activity, most likely caused by the inhibition of NA. Of these, compounds 1, 3, and 6 were highly ranked in shape-focused virtual screening.

Evaluation of three influenza neuraminidase inhibition assays for use in a public health laboratory setting during the 2011-2012 influenza season.

OBJECTIVES: We evaluated the implementation of three commercially available neuraminidase inhibition assays in a public health laboratory (PHL) setting. We also described the drug susceptibility patterns of human influenza A and B circulating in Maryland during the 2011-2012 influenza season. METHODS: From January to May 2012, 169 influenza virus isolates were tested for phenotypic susceptibility to oseltamivir, zanamivir, and peramivir using NA-Fluor(TM), NA-Star®, and NA-XTD(TM) concurrently. A 50% neuraminidase inhibitory concentration (IC50) value was calculated to determine drug susceptibility. We used the standard deviation based on the median absolute deviation of the median analysis to determine the potential for reduced drug susceptibility. We evaluated each assay for the use of resources in high- and low-volume testing scenarios. RESULTS: One of the 25 2009 influenza A (H1N1) pandemic isolates tested was resistant to oseltamivir and peramivir, and sensitive to zanamivir, on all three platforms. Eighty-two influenza A (H3N2) and 62 B isolates were sensitive to all three drugs in all three assays. For a low-volume scenario, NA-Star and NA-XTD took 120 minutes to complete, while NA-Fluor required 300 minutes to complete. The lowest relative cost favored NA-Star. In a high-volume scenario, NA-Fluor had the highest throughput. Reagent use was most efficient when maximizing throughput. Cost efficiency from low- to high-volume testing improved the most for NA-Star. CONCLUSIONS: Our evaluation showed that both chemiluminescent and fluorescent neuraminidase inhibition assays can be successfully implemented in a PHL setting to screen circulating influenza strains for neuraminidase inhibitor resistance. For improved PHL influenza surveillance, it may be essential to develop guidelines for phenotypic drug-resistance testing that take into consideration a PHL's workload and available resources.

Influenza neuraminidase: a druggable target for natural products.

The imminent threat of influenza pandemics and repeatedly reported emergence of new drug-resistant influenza virus strains demonstrate the urgent need for developing innovative and effective antiviral agents for prevention and treatment. At present, influenza neuraminidase (NA), a key enzyme in viral replication, spread, and pathogenesis, is considered to be one of the most promising targets for combating influenza. Despite the substantial medical potential of NA inhibitors (NAIs), only three of these drugs are currently on the market (zanamivir, oseltamivir, and peramivir). Moreover, sudden changes in NAI susceptibility revealed the urgent need in the discovery/identification of novel inhibitors. Nature offers an abundance of biosynthesized compounds comprising chemical scaffolds of high diversity, which present an infinite pool of chemical entities for target-oriented drug discovery in the battle against this highly contagious pathogen. This review illuminates the increasing research efforts of the past decade (2000-2011), focusing on the structure, function and druggability of influenza NA, as well as its inhibition by natural products. Following a critical discussion of publications describing some 150 secondary plant metabolites tested for their inhibitory potential against influenza NA, the impact of three different strategies to identify and develop novel NAIs is presented: (i) bioactivity screening of herbal extracts, (ii) exploitation of empirical knowledge, and (iii) computational approaches. This work addresses the latest developments in theoretical and experimental research on properties of NA that are and will be driving anti-influenza drug development now and in the near future.

Influence of 3-hydroxymethyl xylitol, a novel antidiabetic compound isolated from Casearia esculenta (Roxb.) root, on glycoprotein components in streptozotocin-diabetic rats.

Casearia esculenta root (Roxb.) is widely used in traditional system of medicine to treat diabetes in India. An active compound, 3-hydroxymethyl xylitol (3-HMX), has been isolated, and its optimum dose has been determined in a short duration study and patented. In addition, the long-term effect of 3-HMX in type 2 diabetic rats on antihyperglycemic, antioxidants, antihyperlipidemic, and protein metabolism and kidney marker enzymes was investigated, and its effect was shown previously. In this study, we investigated the effect of 3-HMX on plasma and tissue glycoproteins in streptozotocin-diabetic rats. Animals were divided into five groups viz., control group, 3-HMX (40 mg/kg of body weight) treated group, diabetic group, diabetic+3-HMX (40 mg/kg of body weight), and diabetic+glibenclamide (600 µg/kg of body weight). 3-HMX was administered orally at a dose of 40 mg/kg of body weight for 45 days. The study shows significant increases in the level of sialic acid except kidney and elevated levels of hexose, hexosamine, and fucose in the liver and kidney of diabetic rats, and the treatment with 3-HMX and glibenclamide showed reversal of these parameters toward normalcy. Thus, the study indicates that 3-HMX possesses a significant beneficial effect on glycoprotein components.

Computational studies of influenza A virus at three important targets: hemagglutinin, neuraminidase and M2 protein.

While the seasonal influenza viruses spreading around the world cause the annual epidemics, the recent outbreaks of influenza A virus subtype H5N1 and pandemic H1N1 have raised a global human health concerns. In this review, the applicability of computational techniques focused on three important targets in the viral life cycle: hemagglutinin, neuraminidase and M2 proton channel are summarized. Protein mechanism of action, substrate binding specificity and drug resistance, ligand-target interactions of substrate/inhibitor binding to these three proteins either wild-type or mutant strains are discussed and compared. Advances on the novel anti-influenza agents designed specifically to combat the avian H5N1 and pandemic H1N1 viruses are introduced. A better understanding of molecular inhibition and source of drug resistance as well as a set of newly designed compounds is greatly useful as a rotational guide for synthetic and medicinal chemists to develop a new generation of anti-influenza drugs.

Efficacy of the new neuraminidase inhibitor CS-8958 against H5N1 influenza viruses.

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 microg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants.

N-butyldeoxygalactonojirimycin reduces brain ganglioside and GM2 content in neonatal Sandhoff disease mice.

Sandhoff disease involves the CNS accumulation of ganglioside GM2 and asialo-GM2 (GA2) due to inherited defects in the beta-subunit gene of beta-hexosaminidase A and B (Hexb gene). Accumulation of these glycosphingolipids (GSLs) produces progressive neurodegeneration, ultimately leading to death. Substrate reduction therapy (SRT) aims to decrease the rate of glycosphingolipid (GSL) biosynthesis to compensate for the impaired rate of catabolism. The imino sugar, N-butyldeoxygalactonojirimycin (NB-DGJ) inhibits the first committed step in GSL biosynthesis. NB-DGJ treatment, administered from postnatal day 2 (p-2) to p-5 (600 mg/kg/day)), significantly reduced total brain ganglioside and GM2 content in the Sandhoff disease (Hexb(-/-)) mice, but did not reduce the content of GA2. We also found that NB-DGJ treatment caused a slight, but significant elevation in brain sialidase activity. The drug had no adverse effects on viability, body weight, brain weight, or brain water content in the mice. No significant alterations in neutral lipids or acidic phospholipids were observed in the NB-DGJ-treated Hexb(-/-) mice. Our results show that NB-DGJ is effective in reducing total brain ganglioside and GM2 content at early neonatal ages.

Protective effect of low-concentration chlorine dioxide gas against influenza A virus infection.

Influenza virus infection is one of the major causes of human morbidity and mortality. Between humans, this virus spreads mostly via aerosols excreted from the respiratory system. Current means of prevention of influenza virus infection are not entirely satisfactory because of their limited efficacy. Safe and effective preventive measures against pandemic influenza are greatly needed. We demonstrate that infection of mice induced by aerosols of influenza A virus was prevented by chlorine dioxide (ClO(2)) gas at an extremely low concentration (below the long-term permissible exposure level to humans, namely 0.1 p.p.m.). Mice in semi-closed cages were exposed to aerosols of influenza A virus (1 LD(50)) and ClO(2) gas (0.03 p.p.m.) simultaneously for 15 min. Three days after exposure, pulmonary virus titre (TCID(50)) was 10(2.6+/-1.5) in five mice treated with ClO(2), whilst it was 10(6.7+/-0.2) in five mice that had not been treated (P=0.003). Cumulative mortality after 16 days was 0/10 mice treated with ClO(2) and 7/10 mice that had not been treated (P=0.002). In in vitro experiments, ClO(2) denatured viral envelope proteins (haemagglutinin and neuraminidase) that are indispensable for infectivity of the virus, and abolished infectivity. Taken together, we conclude that ClO(2) gas is effective at preventing aerosol-induced influenza virus infection in mice by denaturing viral envelope proteins at a concentration well below the permissible exposure level to humans. ClO(2) gas could therefore be useful as a preventive means against influenza in places of human activity without necessitating evacuation.

Mangiferin identified in a screening study guided by neuraminidase inhibitory activity.

A screening study on neuraminidase inhibitory constituents was carried out, and activity-guided fractionations of three plants, Gouania obtusifolia, Zizyphus cambodiana, and Mangifera odorata, led to the isolation of eleven compounds (1-11). Mangiferin was identified as a significant neuraminidase inhibitor.

Insulin-like growth factor 1 signaling regulates cytosolic sialidase Neu2 expression during myoblast differentiation and hypertrophy.

Cytosolic sialidase (neuraminidase 2; Neu2) is an enzyme whose expression increases during myoblast differentiation. Here we show that insulin-like growth factor 1 (IGF1)-induced hypertrophy of myoblasts notably increases Neu2 synthesis by activation of the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (P13K/AKT/mTOR) pathway, whereas the proliferative effect mediated by activation of the extracellular regulated kinase 1/2 (ERK1/2) pathway negatively contributed to Neu2 activity. Accordingly, the differentiation L6MLC/IGF-1 cell line, in which the forced postmitotic expression of insulin-like growth factor 1 stimulates a dramatic hypertrophy, was accompanied by a stronger Neu2 increase. Indeed, the hypertrophy induced by transfection of a constitutively activated form of AKT was able to induce high Neu2 activity in C2C12 cells, whereas the transfection of a kinase-inactive form of AKT prevented myotube formation, triggering Neu2 downregulation. Neu2 expression was strictly correlated with IGF-1 signaling also in C2 myoblasts overexpressing the insulin-like growth factor 1 binding protein 5 and therefore not responding to endogenously produced insulin-like growth factor 1. Although Neu2-transfected myoblasts exhibited stronger differentiation, we demonstrated that Neu2 overexpression does not override the block of differentiation mediated by PI3 kinase and mTOR inhibitors. Finally, Neu2 overexpression did not modify the ganglioside pattern of C2C12 cells, suggesting that glycoproteins might be the target of Neu2 activity. Taken together, our data demonstrate that IGF-1-induced differentiation and hypertrophy are driven, at least in part, by Neu2 upregulation and further support the significant role of cytosolic sialidase in myoblasts.

Evolution of the susceptibility to antiviral drugs of A/H3N2 influenza viruses isolated in France from 2002 to 2005.

In 2002, prior to the generalization of zanamivir and oseltamivir utilization in France, we had decided to test a large panel of influenza strains to establish the baseline sensitivity of these viruses to anti-neuraminidase drugs. Since 2002, routine monitoring of resistance relies mainly on the results of neuraminidase enzyme assay. The profile of sensitivity of A/H3N2 viruses has not changed during these three influenza seasons. Neuraminidase N2 was shown to be more sensitive to oseltamivir carboxylate than to zanamivir. Amongst 788 viruses tested, only 10 were found with outliers IC(50) values (seven in 2003, one in 2004, and two in 2005). After the sequencing of the neuraminidase segment of these viruses, seven mutations on the stalk or the head of the glycoprotein (A18S, L23F, C42R, R143V, E199K, S332K, R431N) were detected. Moreover during seasons 2003-2005, 12 additional viruses were characterized by a complete lack of neuraminidase activity. In these viruses, viral resistance to NA inhibitors can be related with the presence of a defective neuraminidase RNA segment as evaluated by a plaque reduction assay, these viruses having a reduced dependence on its neuraminidase activity.