Pandemic 2009 H1N1 Influenza A Virus Carrying a Q136K Mutation in the Neuraminidase Gene Is Resistant to Zanamivir but Exhibits Reduced Fitness in the Guinea Pig Transmission Model

Resistance of influenza A viruses to neuraminidase inhibitors can arise through mutations in the neuraminidase (NA) gene. We show here that a Q136K mutation in the NA of the 2009 pandemic H1N1 virus confers a high degree of resistance to zanamivir. Resistance is accompanied by reduced numbers of NA molecules in viral particles and reduced intrinsic enzymatic activity of mutant NA. Interestingly, the Q136K mutation strongly impairs viral fitness in the guinea pig transmission model.     

Long time scale GPU dynamics reveal the mechanism of drug resistance of the dual mutant I223R/H275Y neuraminidase from H1N1-2009 influenza virus

Multidrug resistance of the pandemic H1N1-2009 strain of influenza has been reported due to widespread treatment using the neuraminidase (NA) inhibitors, oseltamivir (Tamiflu), and zanamivir (Relenza). From clinical data, the single I223R (IR(1)) mutant of H1N1-2009 NA reduced efficacy of oseltamivir and zanamivir by 45 and 10 times, (1) respectively. More seriously, the efficacy of these two inhibitors against the double mutant I223R/H275Y (IRHY(2)) was significantly reduced by a factor of 12?374 and 21 times, respectively, compared to the wild-type.(2) This has led to the question of why the efficacy of the NA inhibitors is reduced by the occurrence of these mutations and, specifically, why the efficacy of oseltamivir against the double mutant IRHY was significantly reduced, to the point where oseltamivir has become an ineffective treatment. In this study, 1 μs of molecular dynamics (MD) simulations was performed to answer these questions. The simulations, run using graphical processors (GPUs), were used to investigate the effect of conformational change upon binding of the NA inhibitors oseltamivir and zanamivir in the wild-type and the IR and IRHY mutant strains. These long time scale dynamics simulations demonstrated that the mechanism of resistance of IRHY to oseltamivir was due to the loss of key hydrogen bonds between the inhibitor and residues in the 150-loop. This allowed NA to transition from a closed to an open conformation. Oseltamivir binds weakly with the open conformation of NA due to poor electrostatic interactions between the inhibitor and the active site. The results suggest that the efficacy of oseltamivir is reduced significantly because of conformational changes that lead to the open form of the 150-loop. This suggests that drug resistance could be overcome by increasing hydrogen bond interactions between NA inhibitors and residues in the 150-loop, with the aim of maintaining the closed conformation, or by designing inhibitors that can form a hydrogen bond to the mutant R223 residue, thereby preventing competition between R223 and R152.     

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.     

Molecular dynamics simulation of oseltamivir resistance in neuraminidase of avian influenza H5N1 virus

The outbreak of avian influenza virus H5N1 has raised a global concern because of its high virulence and mutation rate. Although two classes of antiviral drugs, M2 ion channel protein inhibitors and neuraminidase inhibitors, are expected to be important in controlling the early stages of a potential pandemic. Different strains of influenza viruses have differing degrees of resistance against the antivirals. In order to analyze the detailed information on the viral resistance, molecular dynamics simulations were carried out for the neuraminidase (NA) complex with oseltamivir. The carboxylate of Glu276 of H252Y NA faces toward the O-ethyl-propyl group of oesltamivir, Glu276 of wild-type NA adopts a conformation pointing away from the oesltamivir. τ2 and τ3 torsional angles fluctuation of the oesltamivir are relatively high for the H252Y mutant NA complex. In addition, there are fewer hydrogen bonds between the oesltamivir and H252Y mutation NA. The results show that H252Y mutation NA has high resistance against the drug.     

The binding properties of the H5N1 influenza virus neuraminidase as inferred from molecular modeling

The avian influenza H5N1 virus has emerged as an important pathogen, causing severe disease in humans and posing a pandemic threat. Substrate specificity is crucial for the virus to obtain the ability to spread from avian to human. Therefore, an investigation of the binding properties of ligands at the molecular level is important for understanding the catalytic mechanism of the avian influenza virus neuraminidase and for designing novel and specific inhibitors of H5N1 neuraminidase. Based on the available crystal structure of H5N1, we have characterized the binding properties between sialic acid, methyl 3’sialyllactoside, methyl 6’sialyllactoside and the H5N1 influenza virus neuraminidase using molecular docking and molecular dynamics simulations. Obtained molecular dynamics trajectories were analyzed in terms of ligand conformations, N1-ligand interactions, and in terms of loop flexibility. It was found that in the N1-SA complex the sialic acid ring undergoes a transition from the B _2,5 to the ² C ₅ conformation. However, in the N1-3SL and N1-6SL complexes sialic acid remained in the distorted boat conformation. The obtained results indicate that 3SL has only weak interactions with the 150-loop, whereas the N1-6SL complex shows strong interactions. Most of the differences arise from the various conformations around the glycosidic linkage, between the sialic acid and galactose, which facilitate the above interactions of 6SL with the enzyme, and as a consequence the interactions between the 150- and 430- loops. This finding suggests that the altered flexibility of loops in and around the active site is one of the reasons why the avian N1 preferentially cleaves sialic acid from α-(2-3)-Gal glycoconjugates over α-(2-6)-Gal. These molecular modeling results are consistent with available experimental results on the specificity of N1.     

Helix 4 of the Bacillus thuringiensis Cry1Aa toxin lines the lumen of the ion channel.

The mode of action of Bacillus thuringiensis insecticidal proteins is not well understood. Based on analogies with other bacterial toxins and ion channels, we hypothesized that charged amino acids in helix 4 of the Cry1Aa toxin are critical for toxicity and ion channel function. Using Plutella xylostella as a model target, we analyzed responses to Cry1Aa and eight proteins with altered helix 4 residues. Toxicity was abolished in five charged residue mutants (E129K, R131Q, R131D, D136N, D136C), however, two charged (R127E and R127N) and one polar (N138C) residue mutant retained wild-type toxicity. Compared with Cry1Aa and toxic mutants, nontoxic mutants did not show greatly reduced binding to brush border membrane vesicles, but their ion channel conductance was greatly reduced in planar lipid bilayers. Substituted cysteine accessibility tests showed that in situ restoration of the negative charge of D136C restored conductance to wild-type levels. The results imply that charged amino acids on the Asp-136 side of helix 4 are essential for toxicity and passage of ions through the channel. These results also support a refined version of the umbrella model of membrane integration in which the side of helix 4 containing Asp-136 faces the aqueous lumen of the ion channel.     

Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation

The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.Two classes of antiviral drugs are currently available for the treatment and prophylaxis of influenza, the adamantanes and the neuraminidase (NA) inhibitors (NAIs). The adamantanes were the first agents to be recognized to have anti-influenza virus activities as early as 1964 (2) although the rapid emergence of drug-resistant influenza virus strains has limited their clinical effectiveness (12). The NAIs, zanamivir (Relenza) and oseltamivir (Tamiflu), were the first drugs to be specifically designed as anti-influenza virus agents and have been available on the market in many countries since 1999. During oseltamivir clinical trials, 1 to 4% of treated adults (6) and 5 to 6% of treated children were found to shed resistant viruses (30) although more recent studies have reported resistance in 16 to 18% of viruses from oseltamivir-treated children (20, 29). In contrast to the frequency of resistance seen following oseltamivir treatment, only one occurrence of significant zanamivir resistance has been observed following zanamivir treatment. The zanamivir-resistant strain, an influenza B virus with an R152K NA mutation, was isolated from an immunocompromised patient undergoing prolonged zanamivir treatment (7).In addition to the analysis of influenza viruses isolated from patients undergoing either oseltamivir or zanamivir treatment, surveillance studies that analyze the NAI susceptibility of circulating viruses, predominantly from patients not undergoing NAI treatment, have also been conducted. Studies that have tested viruses isolated prior to the release of the NAIs (1996 to 1999) (23) and after the initiation of clinical use of these drugs (2000 to 2006) (16, 24) have found either no resistance or a very low frequency of resistance. In contrast, analysis of circulating seasonal influenza viruses from Europe during the 2007 to 2008 season revealed that 14% (59/437) of influenza A (H1N1) viruses had significantly decreased susceptibility to oseltamivir (21). Since this initial report, oseltamivir-resistant influenza A (H1N1) strains have spread throughout Europe (11) and have been detected at high frequencies in other countries including the United States (4), Japan (28), South Africa (1) and Oceania and Southeast Asia (17). These influenza A (H1N1) viruses have a mutation of histidine to tyrosine at residue 274 of the NA (N2 NA numbering; residue 275 by N1 NA numbering), which confers a high level of resistance to oseltamivir (10) but has no effect on susceptibility to zanamivir or to the adamantanes.Prior to May 2008, when the oseltamivir-resistant variants became the dominant influenza A (H1N1) strain in Oceania and Southeast Asia (17), NAI sensitivity monitoring conducted at the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, identified a number of influenza A (H1N1) viruses with reduced zanamivir susceptibility. These viruses contained a previously undescribed mutation at residue 136 of the NA. Here, we report on the detection of these mutant viruses from geographically distinct locations, the in vitro and in vivo fitness of the strains, and the finding that the mutant viruses appear to have been preferentially propagated during viral culture in Madin-Darby canine kidney (MDCK) cells.     

Computational studies of H5N1 influenza virus resistance to oseltamivir

Influenza A (H5N1) virus is one of the world's greatest pandemic threats. Neuraminidase (NA) inhibitors, oseltamivir and zanamivir, prevent the spread of influenza, but drug‐resistant viruses have reduced their effectiveness. Resistance depends on the binding properties of NA‐drug complexes. Key residue mutations within the active site of NA glycoproteins diminish binding, thereby resulting in drug resistance. We performed molecular simulations and calculations to characterize the mechanisms of H5N1 influenza virus resistance to oseltamivir and predict potential drug‐resistant mutations. We examined two resistant NA mutations, H274Y and N294S, and one non‐drug‐resistant mutation, E119G. Six‐nanosecond unrestrained molecular dynamic simulations with explicit solvent were performed using NA‐oseltamivir complexes containing either NA wild‐type H5N1 virus or a variant. MM_PBSA techniques were then used to rank the binding free energies of these complexes. Detailed analyses indicated that conformational change of E276 in the Pocket 1 region of NA is a key source of drug resistance in the H274Y mutant but not in the N294S mutant.     

Helix-coil transition of PrP106-126: molecular dynamic study.

A set of 34 molecular dynamic (MD) simulations totaling 305 ns of simulation time of the prion protein-derived peptide PrP106-126 was performed with both explicit and implicit solvent models. The objective of these simulations is to investigate the relative stability of the alpha-helical conformation of the peptide and the mechanism for conversion from the helix to a random-coil structure. At neutral pH, the wild-type peptide was found to lose its initial helical structure very fast, within a few nanoseconds (ns) from the beginning of the simulations. The helix breaks up in the middle and then unwinds to the termini. The spontaneous transition into the random coil structure is governed by the hydrophobic interaction between His(111) and Val(122). The A117V mutation, which is linked to GSS disease, was found to destabilize the helix conformation of the peptide significantly, leading to a complete loss of helicity approximately 1 ns faster than in the wild-type. Furthermore, the A117V mutant exhibits a different mechanism for helix-coil conversion, wherein the helix begins to break up at the C-terminus and then gradually to unwind towards the N-terminus. In most simulations, the mutation was found to speed up the conversion through an additional hydrophobic interaction between Met(112) and the mutated residue Val(117), an interaction that did not exist in the wild-type peptide. Finally, the beta-sheet conformation of the wild-type peptide was found to be less stable at acidic pH due to a destabilization of the His(111)-Val(122), since at acidic pH this histidine is protonated and is unlikely to participate in hydrophobic interaction.     

Locking the 150-Cavity Open: In Silico Design and Verification of Influenza Neuraminidase Inhibitors

Neuraminidase (NA) of influenza is a key target for virus infection control and the recently discovered open 150-cavity in group-1 NA provides new opportunity for novel inhibitors design. In this study, we used a combination of theoretical methods including fragment docking, molecular linking and molecular dynamics simulations to design ligands that specifically target at the 150-cavity. Through in silico screening of a fragment compound library on the open 150-cavity of NA, a few best scored fragment compounds were selected to link with Zanamivir, one NA-targeting drug. The resultant new ligands may bind both the active site and the 150-cavity of NA simultaneously. Extensive molecular dynamics simulations in explicit solvent were applied to validate the binding between NA and the designed ligands. Moreover, two control systems, a positive control using Zanamivir and a negative control using a low-affinity ligand 3-(p-tolyl) allyl-Neu5Ac2en (ETT, abbreviation reported in the PDB) found in a recent experimental work, were employed to calibrate the simulation method. During the simulations, ETT was observed to detach from NA, on the contrary, both Zanamivir and our designed ligand bind NA firmly. Our study provides a prospective way to design novel inhibitors for controlling the spread of influenza virus.     

Neuraminidase inhibitor R-125489--a promising drug for treating influenza virus: steered molecular dynamics approach

Two neuraminidase inhibitors, oseltamivir and zanamivir, are important drug treatments for influenza. Oseltamivir-resistant mutants of the influenza virus A/H1N1 and A/H5N1 have emerged, necessitating the development of new long-acting antiviral agents. One such agent is a new neuraminidase inhibitor R-125489 and its prodrug CS-8958. An atomic level understanding of the nature of this antiviral agents binding is still missing. We address this gap in our knowledge by applying steered molecular dynamics (SMD) simulations to different subtypes of seasonal and highly pathogenic influenza viruses. We show that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. Based on results obtained by SMD and the molecular mechanics-Poisson–Boltzmann surface area method, we predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus as its binding affinity does not vary much across these systems. The high correlation level between theoretically determined rupture forces and experimental data on binding energies for the large number of systems studied here implies that SMD is a promising tool for drug design.     

Comparison of Mutation Patterns in Full-Genome A/H3N2 Influenza Sequences Obtained Directly from Clinical Samples and the Same Samples after a Single MDCK Passage

Human influenza viruses can be isolated efficiently from clinical samples using Madin-Darby canine kidney (MDCK) cells. However, this process is known to induce mutations in the virus as it adapts to this non-human cell-line. We performed a systematic study to record the pattern of MDCK-induced mutations observed across the whole influenza A/H3N2 genome. Seventy-seven clinical samples collected from 2009-2011 were included in the study. Two full influenza genomes were obtained for each sample: one from virus obtained directly from the clinical sample and one from the matching isolate cultured in MDCK cells. Comparison of the full-genome sequences obtained from each of these sources showed that 42% of the 77 isolates had acquired at least one MDCK-induced mutation. The presence or absence of these mutations was independent of viral load or sample origin (in-patients versus out-patients). Notably, all the five hemagglutinin missense mutations were observed at the hemaggutinin 1 domain only, particularly within or proximal to the receptor binding sites and antigenic site of the virus. Furthermore, 23% of the 77 isolates had undergone a MDCK-induced missense mutation, D151G/N, in the neuraminidase segment. This mutation has been found to be associated with reduced drug sensitivity towards the neuraminidase inhibitors and increased viral receptor binding efficiency to host cells. In contrast, none of the neuraminidase sequences obtained directly from the clinical samples contained the D151G/N mutation, suggesting that this mutation may be an indicator of MDCK culture-induced changes. These D151 mutations can confound the interpretation of the hemagglutination inhibition assay and neuraminidase inhibitor resistance results when these are based on MDCK isolates. Such isolates are currently in routine use in the WHO influenza vaccine and drug-resistance surveillance programs. Potential data interpretation miscalls can therefore be avoided by careful exclusion of such D151 mutants after further sequence analysis.     

Protein-protein interactions in actin-myosin binding and structural effects of R405Q mutation: a molecular dynamics study

Detailed residue-wise interactions involved in the binding of myosin to actin in the rigor conformation without nucleotides have been examined using molecular dynamics simulations of the chicken skeletal myosin head complexed with two actin monomers, based on the cryo-microscopic model of Holmes et al. (Nature 2003;425:423-427). The overall interaction is largely electrostatic in nature, because of the charged residues in the four loops surrounding the central primary binding site. The 50k/20k loop, disordered in crystal structures and in simulations of free myosin in solution, was found to be in a conformation stabilized with 1 - 2 internal salt bridges. The cardiomyopathy loop forms 2 - 3 interprotein salt bridges with actin monomers upon binding, whereas its Arg405 residue, the mutation site associated with the hypertrophic cardiomyopathy, forms a strong salt bridge with Glu605 in the neighboring helix away from actin in the actin-bound myosin. The myopathy loop of the R405Q mutant maintains a high degree of two-strand beta-sheet character when bound to actin with the corresponding salt bridges broken.     

The DRY motif as a molecular switch of the human oxytocin receptor

The human oxytocin receptor is known to exhibit promiscuous activity by coupling to both Galpha(q) and Galpha(i) G proteins to activate distinct signaling pathways. A single-amino acid substitution within the highly conserved E/DRY motif at the cytosolic extension of helix 3 [i.e., D136(3.49)N] increased the rate of both basal and agonist-stimulated inositol phosphate (IP(3)) accumulation of the receptor. Furthermore, like for a typical constitutively active receptor, the partial agonist arginine vasopressin behaved as a full agonist for the D136(3.49)N mutant. Subsequently, both oxytocin and arginine vasopressin showed an increased potency in stimulating IP3 accumulation as compared to the wild-type receptor. Very interestingly, our experiments provide strong evidence that the D136(3.49)N mutant inhibits receptor signaling via Galpha(i)-mediated pathways while increasing the activity through the Galpha(q)-mediated pathways. Molecular simulations of the free and OT-bound forms of wild-type OTR and of the D136(3.49)N constitutively active mutant suggest that the receptor portions close to the E/DRY and NPxxY motifs are particularly susceptible to undergoing structural modification in response to activating mutations and agonist binding. Furthermore, computational modeling suggests that the OT-bound form of wild-type OTR is able to explore more states than the OT-bound form of the D136(3.49)N constitutively active mutant, consistent with its G protein promiscuity. Taken together, these observations emphasize the important role of the E/DRY motif not only in receptor activation but also in the promiscuity of G protein coupling. Knowledge of the mechanism of selective G protein coupling could aid drug discovery efforts to identify signaling specific therapies.     

Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.     

In silico analysis of drug-resistant mutant of neuraminidase (N294S) against oseltamivir

The recent H1N1 influenza pandemic has attracted worldwide attention due to the high infection rate. Oseltamivir is a new class of anti-viral agent approved for the treatment and prevention of influenza infections. The principal target for this drug is a virus surface glycoprotein, neuraminidase (NA), which facilitates the release of nascent virus and thus spreads infection. Until recently, only a low prevalence of neuraminidase inhibitor (NAI) resistance (<1 %) had been detected in circulating viruses. However, there have been reports of significant numbers of A (H1N1) influenza strains with a N294S neuraminidase mutation that was highly resistant to the NAI, oseltamivir. Hence, in the present study, we highlight the effect of point mutation-induced oseltamivir resistance in H1N1 subtype neuraminidases by molecular simulation approach. The docking analysis reveals that mutation (N294S) significantly affects the binding affinity of oseltamivir with mutant type NA. This is mainly due to the decrease in the flexibility of binding site residues and the difference in prevalence of hydrogen bonds in the wild and mutant structures. This study throws light on the possible effects of drug-resistant mutations on the large functionally important collective motions in biological systems.     

In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675

With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.     

Detection of resistance mutations to antivirals oseltamivir and zanamivir in avian influenza A viruses isolated from wild birds

The neuraminidase (NA) inhibitors oseltamivir and zanamivir are the first-line of defense against potentially fatal variants of influenza A pandemic strains. However, if resistant virus strains start to arise easily or at a high frequency, a new anti-influenza strategy will be necessary. This study aimed to investigate if and to what extent NA inhibitor–resistant mutants exist in the wild population of influenza A viruses that inhabit wild birds. NA sequences of all NA subtypes available from 5490 avian, 379 swine and 122 environmental isolates were extracted from NCBI databases. In addition, a dataset containing 230 virus isolates from mallard collected at Ottenby Bird Observatory (Öland, Sweden) was analyzed. Isolated NA RNA fragments from Ottenby were transformed to cDNA by RT-PCR, which was followed by sequencing. The analysis of genotypic profiles for NAs from both data sets in regard to antiviral resistance mutations was performed using bioinformatics tools. All 6221 sequences were scanned for oseltamivir- (I117V, E119V, D198N, I222V, H274Y, R292K, N294S and I314V) and zanamivir-related mutations (V116A, R118K, E119G/A/D, Q136K, D151E, R152K, R224K, E276D, R292K and R371K). Of the sequences from the avian NCBI dataset, 132 (2.4%) carried at least one, or in two cases even two and three, NA inhibitor resistance mutations. Swine and environmental isolates from the same data set had 18 (4.75%) and one (0.82%) mutant, respectively, with at least one mutation. The Ottenby sequences carried at least one mutation in 15 cases (6.52%). Therefore, resistant strains were more frequently found in Ottenby samples than in NCBI data sets. However, it is still uncertain if these mutations are the result of natural variations in the viruses or if they are induced by the selective pressure of xenobiotics (e.g., oseltamivir, zanamivir).