Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses

An epidemic of an avian-origin H7N9 influenza virus has recently emerged in China, infecting 134 patients of which 45 have died. This is the first time that an influenza virus harboring an N9 serotype neuraminidase (NA) has been known to infect humans. H7N9 viruses are divergent and at least two distinct NAs and hemagglutinins (HAs) have been found, respectively, from clinical isolates. The prototypes of these viruses are A/Anhui/1/2013 and A/Shanghai/1/2013. NAs from these two viruses are distinct as the A/Shanghai/1/2013 NA has an R294K substitution that can confer NA inhibitor oseltamivir resistance. Oseltamivir is by far the most commonly used anti-influenza drug due to its potency and high bioavailability. In this study, we show that an R294K substitution results in multidrug resistance with extreme oseltamivir resistance (over 100 000-fold) using protein- and virus-based assays. To determine the molecular basis for the inhibitor resistance, we solved high-resolution crystal structures of NAs from A/Anhui/1/2013 N9 (R294-containing) and A/Shanghai/1/2013 N9 (K294-containing). R294K substitution results in an unfavorable E276 conformation for oseltamivir binding, and consequently loss of inhibitor carboxylate interactions, which compromises the binding of all classical NA ligands/inhibitors. Moreover, we found that R294K substitution results in reduced NA catalytic efficiency along with lower viral fitness. This helps to explain why K294 has predominantly been found in clinical cases of H7N9 infection under the selective pressure of oseltamivir treatment and not in the dominant human-infecting viruses. This implies that oseltamivir can still be efficiently used in the treatment of H7N9 infections.     

Applications of the ArgusLab4/AScore protocol in the structure-based binding affinity prediction of various inhibitors of group-1 and group-2 influenza virus neuraminidases (NAs)

Using the crystal structures of inhibitors bound to either group-2 or group-1 neuraminidases (NAs), AScore/ShapeDock (GaDock) scoring was shown to identify the binding modes in agreement with the experiment for all inhibitors docked in their own NA/inhibitor crystal structures. To investigate the effect of small changes in protein structure on predicted binding modes, in a set of 132 docking experiments (11 inhibitors docked in 12 group-2 NA structures), AScore/ShapeDock (GaDock) identified the correct binding modes of 116 complexes. In a total of 88 docking experiments (8 inhibitors docked in 11 group-1 NA structures), AScore/ShapeDock predicted 80 binding modes correctly. Flexible AScore/ShapeDock docking, as quite reproducible, is suggested to be convenient for designing novel H5N1 inhibitors.     

Study of drug resistance of chicken influenza A virus (H5N1) from homology-modeled 3D structures of neuraminidases

The spread of highly pathogenic H5N1 influenza virus in many Asian and European countries as well as its drug-resistance have raised serious worldwide concerns. In this paper, the structure-activity relationship between NA (neuraminidase) and its three inhibitors (DANA, zanamivir, and oseltamivir) was investigated. A homology model of H5N1-NA (BAE46950), which is the first reported oseltamivir-resistance virus strain, and the 108 homology-modeled 3D structures of chicken influenza H5N1 NAs downloaded from the website at , formed the molecular structural basis for the drug-resistance study. The multiple sequence and structure alignment for these NAs indicated that 11 functional residues were highly conserved except for AAF02313 with the mutated virus strain. However, the framework residues have remarkable mutations from N9-NA to H5N1-NA, and a few mutated residues were observed in different H5N1-NAs. A partially hydrophobic site S5 (formed by Ala246 and Thr247) in N9-NA is changed to a hydrophilic site (formed by Ala227 and Asn228) in H5N1-NA, while a hydrophilic site S6 (formed by Asn346 and Asn347) in N9-NA was replaced by a hydrophobic site (formed by Ala323 and Tyr324). All these mutations might be the reason for the oseltamivir-resistance by some H5N1 viruses. In order to find the possible drug-resistant H5N1 virus, similarity analysis was performed using the BAE46950 sequence as the benchmark template, and 21 sequences were found from the database of the 108 H5N1 NAs that had over 95% sequence similarity with BAE46950.     

Plasticity of 150-Loop in Influenza Neuraminidase Explored by Hamiltonian Replica Exchange Molecular Dynamics Simulations

Neuraminidase (NA) of influenza is a key target for antiviral inhibitors, and the 150-cavity in group-1 NA provides new insight in treating this disease. However, NA of 2009 pandemic influenza (09N1) was found lacking this cavity in a crystal structure. To address the issue of flexibility of the 150-loop, Hamiltonian replica exchange molecular dynamics simulations were performed on different groups of NAs. Free energy landscape calculated based on the volume of 150-cavity indicates that 09N1 prefers open forms of 150-loop. The turn A (residues 147–150) of the 150-loop is discovered as the most dynamical motif which induces the inter-conversion of this loop among different conformations. In the turn A, the backbone dynamic of residue 149 is highly related with the shape of 150-loop, thus can function as a marker for the conformation of 150-loop. As a contrast, the closed conformation of 150-loop is more energetically favorable in N2, one of group-2 NAs. The D147-H150 salt bridge is found having no correlation with the conformation of 150-loop. Instead the intimate salt bridge interaction between the 150 and 430 loops in N2 variant contributes the stabilizing factor for the closed form of 150-loop. The clustering analysis elaborates the structural plasticity of the loop. This enhanced sampling simulation provides more information in further structural-based drug discovery on influenza virus.     

Neuraminidase hemadsorption activity, conserved in avian influenza A viruses, does not influence viral replication in ducks.

The N1 and N9 neuraminidase (NA) subtypes of influenza A viruses exhibit significant hemadsorption activity that localizes to a site distinct from that of the enzymatic active site. To determine the conservation of hemadsorption activity among different NAs, we have examined most of the NA subtypes from avian, swine, equine, and human virus isolates. All subtypes of avian virus NAs examined and one equine virus N8 NA possessed high levels of hemadsorption activity. A swine virus N1 NA exhibited only weak hemadsorption activity, while in human virus N1 and N2 NAs, the activity was detected at a much lower level than in avian virus NAs. NAs which possessed hemadsorption activity for chicken erythrocytes (RBCs) were similarly able to adsorb human RBCs. However, none of the hemadsorption-positive NAs could bind equine, swine, or bovine RBCs, suggesting that RBCs from these species lack molecules, recognized by the NA hemadsorption site, present on human and chicken RBCs. Mutagenesis of the putative hemadsorption site of A/duck/Hong Kong/7/75 N2 NA abolished the high level of hemadsorption activity exhibited by the wild-type protein but also resulted in a 50% reduction of the NA enzymatic activity. A transfectant virus, generated by reverse genetics, containing this mutated NA replicated 10-fold less efficiently in chicken embryo fibroblast cultures than did a transfectant virus expressing the wild-type NA. However, both viruses replicated equally well in Peking ducks. Although conservation of NA hemadsorption activity among avian virus NAs suggests the maintenance of a required function of NA, loss of the activity does not preclude the replication of the virus in an avian host.     

Fusion-Related Host Proteins Are Actively Regulated by NA during Influenza Infection as Revealed by Quantitative Proteomics Analysis

Three recombinant influenza A viruses with different neuraminidases (NAs) in the background of A/PR/8/34 (PR8), named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine) viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK) cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS) and two-dimensional gel electrophoresis (2-DE), we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses.     

Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure

Background

Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied.

Aim and Methods

In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naïve Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype.

Results and Conclusion

Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.     

Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses

To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>dimer. AH N1 from pseudoparticles existed in the forms monomer>dimer, but after insertion of 09s60, it existed in the forms dimer>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.     

Insights from investigating the interaction of oseltamivir (Tamiflu) with neuraminidase of the 2009 H1N1 swine flu virus

The neuraminidase (NA) of influenza virus is the target of anti-flu drugs oseltamivir and zanamivir. Clinical practices showed that oseltamivir was effective to treat the 2009-H1N1 influenza but failed to the 2006-H5N1 avian influenza. To perform an in-depth analysis on such a drug-resistance problem, the 2009-H1N1-NA structure was developed. To compare it with the crystal 2006-H5N1-NA structure as well as the 1918 influenza virus H1N1-NA structure, the multiple sequential and structural alignments were performed. It has been revealed that the hydrophobic residue Try347 in H5N1-NA does not match with the hydrophilic carboxyl group of oseltamivir as in the case of H1N1-NA. This may be the reason why H5N1 avian influenza virus is drug-resistant to oseltamivir. The finding provides useful insights for how to modify the existing drugs, such as oseltamivir and zanamivir, making them not only become more effective against H1N1 virus but also effective against H5N1 virus.     

A generic system for the expression and purification of soluble and stable influenza neuraminidase

The influenza surface glycoprotein neuraminidase (NA) is essential for the efficient spread of the virus. Antiviral drugs such as Tamiflu (oseltamivir) and Relenza (zanamivir) that inhibit NA enzyme activity have been shown to be effective in the treatment of influenza infections. The recent 'swine flu' pandemic and world-wide emergence of Tamiflu-resistant seasonal human influenza A(H1N1) H(274)Y have highlighted the need for the ongoing development of new anti-virals, efficient production of vaccine proteins and novel diagnostic tools. Each of these goals could benefit from the production of large quantities of highly pure and stable NA. This publication describes a generic expression system for NAs in a baculovirus Expression Vector System (BEVS) that is capable of expressing milligram amounts of recombinant NA. To construct NAs with increased stability, the natural influenza NA stalk was replaced by two different artificial tetramerization domains that drive the formation of catalytically active NA homotetramers: GCN4-pLI from yeast or the Tetrabrachion tetramerization domain from Staphylothermus marinus. Both recombinant NAs are secreted as FLAG-tagged proteins to allow for rapid and simple purification. The Tetrabrachion-based NA showed good solubility, increased stability and biochemical properties closer to the original viral NA than the GCN4-pLI based construct. The expressed quantities and high quality of the purified recombinant NA suggest that this expression system is capable of producing recombinant NA for a broad range of applications including high-throughput drug screening, protein crystallisation, or vaccine development.     

Functional and Structural Analysis of Influenza Virus Neuraminidase N3 Offers Further Insight into the Mechanisms of Oseltamivir Resistance

The influenza virus neuraminidase H274Y substitution is a highly prevalent amino acid substitution associated with resistance to the most heavily used influenza drug, oseltamivir. Previous structural studies suggest that the group specific 252 residue (Y252 in group 1 and T252 in group 2) might be a key factor underlying H274Y resistance. However, H274Y has only been reported in N1 subtypes, which indicates that there must be additional key residues that determine H274Y resistance. Furthermore, we found that members of NA serotype N3 also possess Y252, raising the key question as to whether or not H274Y resistance may also be possible for some group 2 NAs. Here, we demonstrate that the H274Y substitution results in mild oseltamivir resistance for N3. Comparative structural analysis of N3, N1, and their 274Y variants indicates that the interaction of residue 296 (H in N1 and nonaromatic for other serotypes) with conserved W295 is another important determinant of oseltamivir resistance.     

Structure-activity relationship of flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities

Flavonoids are polyphenolic compounds that widely exist in plant kingdom, and the structure-activity relationship (SAR) of 25 flavonoids was studied on neuraminidase (NA) activity of influenza virus. Three typical influenza virus strains A/PR/8/34 (H1N1), A/Jinan/15/90 (H3N2), and B/Jiangshu/10/2003 were used as the source of NAs, the average of IC(50)s of these compounds on these NAs was used in the SAR analysis. The order of potency for NA inhibition was as follows: aurones>flavon(ol)es>isoflavones>flavanon(ol)es and flavan(ol)es. The SAR analysis of flavonoids on influenza virus NAs revealed that for good inhibitory effect, the 4'-OH, 7-OH, C4O, and C2C3 functionalities were essential, and the presence of a glycosylation group greatly reduced NA inhibition. The in vitro anti-viral activities of eight flavonoids were evaluated using a cytopathic effect (CPE) reduction method, the assay results confirmed the SAR as influenza virus neuraminidase inhibitors. The findings of this study provide important information for the exploitation and utilization of flavonoids as NA inhibitors for influenza treatment.     

Outside‐binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1

The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA‐oseltamivir complex (PDB ID: 3NSS) was used as a wild‐type structure. After selecting the target NA sequences, their three‐dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free‐energy analysis using the MM‐PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM‐PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature. © 2012 Wiley Periodicals, Inc.     

Oseltamivir-resistant influenza A viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol

Influenza viruses resistant to the neuraminidase (NA) inhibitor oseltamivir arise under drug selection pressure both in vitro and in vivo. Several mutations in the active site of the viral NA are known to confer relative resistance to oseltamivir, and influenza viruses with certain oseltamivir resistance mutations have been shown to transmit efficiently among cocaged ferrets. However, it is not known whether NA mutations alter aerosol transmission of drug-resistant influenza virus. Here, we demonstrate that recombinant human influenza A/H3N2 viruses without and with oseltamivir resistance mutations (in which NA carries the mutation E119V or the double mutations E119V I222V) have similar in ovo growth kinetics and infectivity in guinea pigs. These viruses also transmit efficiently by the contact route among cocaged guinea pigs, as in the ferret model. However, in an aerosol transmission model, in which guinea pigs are caged separately, the oseltamivir-resistant viruses transmit poorly or not at all; in contrast, the oseltamivir-sensitive virus transmits efficiently even in the absence of direct contact. The present results suggest that oseltamivir resistance mutations reduce aerosol transmission of influenza virus, which could have implications for public health measures taken in the event of an influenza pandemic.     

Influenza A virus N5 neuraminidase has an extended 150-cavity

There are 9 serotypes of neuraminidase (NA) from influenza A virus (N1 to N9), which are classified into two groups based on primary sequences (groups 1 and 2). The structural hallmark of the two groups is the presence or absence of an extra 150-cavity (formed by the 150-loop) in the active site. Thus far, structures of NAs from 6 out of the 9 serotypes have been solved. Here, we solved the N5 structure, the last unknown structure group 1 serotype with a unique Asn147 residue in its 150-loop, demonstrating that it has an extended 150-cavity that closes upon inhibitor binding.     

Analogue inhibitors by modifying oseltamivir based on the crystal neuraminidase structure for treating drug-resistant H5N1 virus

The worldwide spread of H5N1 avian influenza and the increasing reports about its resistance to the existing drugs have made a priority for the development of the new anti-influenza molecules. The crystal structure of H5N1 avian influenza neuraminidase reported recently by Russell et al. [R.J. Russell, L.F. Haire, D.J. Stevens, P.J. Collins, Y. P. Lin, G.M. Blackburn, A.J. Hay, S.J. Gamblin, J.J. Skehel, The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design, Nature 443 (2006) 45-49] have provided new opportunities for drug design in this regard. It is revealed through the structure that the active sites of the group-1 neuraminidases, which contain the N1 subtype, have a very different three-dimensional structure from those of group-2 neuraminidases. The key difference is in the 150-loop cavity adjacent to the conserved active site in neuraminidase. Based on these findings and by modifying oseltamivir, six analog inhibitors were proposed as candidates for developing inhibitors against H5N1 virus, particularly against the oseltamivir-resistant H5N1 virus strain.     

Glycan binding and specificity of viral influenza neuraminidases by classical molecular dynamics and replica exchange molecular dynamics simulations

Two important glycoproteins on the influenza virus membrane, hemagglutinin (HA) and neuraminidase (NA), are relevant to virus replication. As previously reported, HA has a substrate specificity towards SIA-2,3-GAL-1,4-NAG (3SL) and SIA-2,6-GAL-1,4-NAG (6SL) glycans, while NA can cleave both types of linkages. However, the substrate binding into NA and its preference are not well understood. In this work, the glycan binding and specificity of human and avian NAs were evaluated by classical molecular dynamics (MD) simulations, whilst the conformational diversity of 3SL avian and 6SL human glycans in an unbound state was investigated by replica exchange MD simulations. The results indicated that the 3SL avian receptor fits well in the binding cavity of all NAs and does not require a conformational change for such binding compared to the flexible shape of the 6SL human receptor. From the QM/MM-GBSA binding free energy and decomposition free energy data, 6SL showed a much stronger binding towards human NAs (H1N1, H2N2 and H3N2) than to avian NAs (H5N1 and H7N9). This suggests that influenza NAs have a substrate specificity corresponding to their HA, indicating the functional balance between the two important glycoproteins. Both linkages show distinct glycan topologies when complexed with NAs, while the flexibility of torsion angles between GAL and NAG in 6SL results in the various shapes of glycan and different binding patterns. Lower conformational diversities of both glycans when bound to NA compared to the unbound state were found, and were required in order to be accommodated within the NA cavity.

Communicated by Ramaswamy H. Sarma     

Structural characterization of the 1918 influenza virus H1N1 neuraminidase

Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 "Spanish flu" (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-A and 1.45-A resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147 to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.     

Discovery of acylguanidine oseltamivir carboxylate derivatives as potent neuraminidase inhibitors

In search of novel anti-influenza agents with higher potency, a series of acylguanidine oseltamivir carboxylate analogues were synthesized and evaluated against influenza viruses (H1N1 and H3N2) in vitro. The representative compounds with strong inhibitory activities (IC₅₀ <40 nM) against neuraminidase (NA) were further tested against the NA from oseltamivir-resistant strain (H259Y). Among them, compounds 9 and 17 were potent NA inhibitors that exhibited a 5 and 11-fold increase in activity comparing with oseltamivir carboxylate (2, OC) against the H259Y mutant, respectively. Furthermore, the effect against influenza virus H259Y mutant (H1N1) replication and cytotoxicity assays indicated that compounds 9 and 17 exhibited a 20 and 6-fold increase than the parent compound 2, and had no obvious cytotoxicity in vitro. Moreover, the molecular docking studies revealed that the docking modes of compounds 9 and 17 were different from that of oseltamivir, and the new hydrogen bonds and hydrophobic interaction were formed in this case. This work provided unique insights in the discovery of potent inhibitors against NAs from wild-type and oseltamivir-resistant strains.     

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 µg/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.