N1 Subtype Research Articles

Presence of Serum Antibodies to Influenza A Subtypes H5 and N1 in Swans and Ibises in French Wetlands, Irrespective of Highly Pathogenic H5N1 Natural Infection

Highly pathogenic (HP) avian influenza A viruses (AIVs) subtype H5N1 (subclade 2.2) were detected in wild birds during outbreaks in France during winter 2006 and summer 2007 in la Dombes wetlands (eastern France) and in Moselle wetlands (northeastern France), respectively. Blood samples from apparently healthy wild birds were collected in 2006 and 2007 from the end of the outbreak to several weeks after the influenza A outbreak inside and outside the contaminated areas, and in 2008 outside the contaminated areas. The samples were tested for the presence and/or quantitation of serum antibodies to influenza A subtypes H5 and N1 using hemagglutination inhibition tests (HITs), a commercial N1-specific enzyme-linked immunosorbent assay kit, and virus neutralization assay. In the HIT, low pathogenicity (LP) and HP H5 AIVs (belonging to H5N1, H5N2, and H5N3 subtypes) were used as antigens. One hundred mute swans were bled in the la Dombes outbreak area in 2006. During 2007, 46 mallards, 69 common pochards, and 59 mute swans were sampled in the Moselle outbreak area. For comparison, blood samples were also collected in 2007 from 60 mute swans from the Marne department where no HP H5N1 influenza A cases have been reported, and in 2008 from 111 sacred ibises in western France where no HP H5N1 influenza A infections in wild birds have been reported either. Mute swans (irrespective of their origin and time of sampling) and sacred ibises (from an area with no known outbreaks) had the highest prevalence of positive sera in the H5 HIT (49-69% and 64%, respectively). The prevalence of anti-H5 antibodies in mallards and common pochards was lower (28% and 27%, respectively). Positive H5- and N1-antibody responses were also significantly associated in swans (irrespective of their origin and time of sampling) and in sacred ibises. However, in swans from the area without outbreaks, the HIT titer against an H5N1 LPAIV was significantly higher than against an H5N1 2.2.1 HPAIV, whereas no difference could be shown for swans from the outbreak areas sampled in 2006 and 2007. These results suggest that ibises and swans from areas without declared outbreaks had acquired humoral immunity after AIV infections with subtypes H5 and N1 but independently from HP H5N1 infection. However, for swans living in outbreak areas, it cannot be excluded that this immunity might result from either a subclinical or a nonlethal infection by HP H5N1.

A Heterologous Neuraminidase Subtype Strategy for the Differentiation of Infected and Vaccinated Animals (DIVA) for Avian Influenza Virus Using an Alternative Neuraminidase Inhibition Test

The option of vaccinating poultry against avian influenza (AI) as a control tool is gaining greater acceptance by governments and the poultry industry worldwide. One disadvantage about vaccination with killed whole-virus vaccines is the resulting inability to use common serologic diagnostic tests for surveillance to identify infected flocks. There has been considerable effort to develop a reliable test for the differentiation of infected from vaccinated animals (DIVA). The heterologous neuraminidase (NA) subtype DIVA approach has been used with some success in the field accompanied by an ad hoc serologic test. The traditional NA inhibition (NI) test can be used for all nine NA subtypes, but it is time consuming, and it is not designed to screen large numbers of samples. In this study, a quantitative NI test using MUN (2'-[4-methylumbelliferyl]-alpha-D-Nacetylneuraminic acid sodium salt hydrate) as an NA substrate was investigated as an alternative to the traditional fetuin-based NI test in a heterologous neuraminidase DIVA strategy. Serum NI activity was determined in chickens administered different vaccines containing different H5 and NA subtypes and challenged with a highly pathogenic avian influenza (HPAI) H5N2 virus. Prior to challenge, the NI DIVA test clearly discriminated between chickens receiving vaccines containing different antigens (e.g., N8 or N9) from control birds that had no NA antibody. Some birds began to seroconvert 1 wk postchallenge, and 100% of the vaccinated birds had significant levels of N2 NI activity. This activity did not interfere with the presence of vaccine-induced NI activity against N8 or N9 subtypes. The level of N2-specific NI activity continued to increase to the last sampling date, 4 wk postchallenge, indicating the potential use for the heterologous NA-based DIVA strategy in the field.

Presence of Serum Antibodies to Influenza a Subtypes H5 and N1 in Swans and Ibises in French Wetlands, Irrespective of Highly Pathogenic H5N1 Natural Infection

Presence of Serum Antibodies to Influenza a Subtypes H5 and N1 in Swans and Ibises in French Wetlands, Irrespective of Highly Pathogenic H5N1 Natural Infection

Development and Evaluation of an Avian Influenza, Neuraminidase Subtype 1, Indirect Enzyme-Linked Immunosorbent Assay for Poultry Using the Differentiation of Infected from Vaccinated Animals Control Strategy

An indirect enzyme-linked immunosorbent assay (ELISA) was developed using baculovirus, purified, recombinant N1 protein from A/chicken/Indonesia/PA7/2003 (H5N1) virus. The N1-ELISA showed high selectivity for detection of N1 antibodies, with no cross-reactivity with other neuraminidase subtypes, and broad reactivity with sera to N1 subtype isolates from North American and Eurasian lineages. Sensitivity of the N1-ELISA to detect N1 antibodies in turkey sera, collected 3 wk after H1N1 vaccination, was comparable to detection of avian influenza antibodies by the commercial, indirect ELISAs ProFLOK AIV Plus ELISA Kit (Synbiotics, Kansas City, MO) and Avian Influenza Virus Antibody Test Kit (IDEXX, Westbrook, ME). However, 6 wk after vaccination, the Synbiotics ELISA kit performed better than the N1-ELISA and the IDEXX ELISA kit. An evaluation was made of the ability of the N1-ELISA to discriminate vaccinated chickens from subsequently challenged chickens. Two experiments were conducted, chickens were vaccinated with inactivated H5N2 and H5N9 viruses and challenged with highly pathogenic H5N1 virus, and chickens were vaccinated with recombinant poxvirus vaccine encoding H7 and challenged with highly pathogenic H7N1 virus. Serum samples were collected at 14 days postchallenge and tested by hemagglutination inhibition (HI), quantitative neuraminidase inhibition (NI), and N1-ELISA. At 2 days postchallenge, oropharyngeal swabs were collected for virus isolation (VI) to confirm infection. The N1-ELISA was in fair agreement with VI and HI results. Although the N1-ELISA showed a lower sensitivity than the NI assay, it was demonstrated that detection of N1 antibodies by ELISA was an effective and rapid assay to identify exposure to the challenge virus in vaccinated chickens. Therefore, N1-ELISA can facilitate a vaccination strategy with differentiation of infected from vaccinated animals using a neuraminidase heterologous approach.

Combinatorial Design of Avian Influenza Neuraminidase Inhibitors Containing Pyrrolidine Core with a Reduced Susceptibility to Viral Drug Resistance

Using computer-assisted combinatorial chemistry techniques, we have designed a virtual library of anti-influenza agents, analogs of inhibitor A-315675, containing a novel pyrrolidine core, which effectively inhibits both wild type and common oseltamivir-resistant mutant forms of the neuraminidase (NA) subtype N1 of avian influenza virus H5N1. A target-specific Potential of Mean Force (PMF) scoring function parameterized on a training set of 13 known pyrrolidine-based inhibitors of NA and validated on 3 others was used to predict the N1 inhibition constants for the focused library of A-315675 analogs. Nine virtual hits (best pyrrolidine inhibitors designed in the present study) are predicted to exhibit inhibition constants in the low picomolar range, up to 200 fold lower than the parent inhibitor A-315675 while displaying favorable predicted ADME-related properties. Proposed small highly-focused combinatorial subsets composed of R-groups most frequently occurring in the 200 most active analogs can be useful as a guide for synthetic and medicinal chemists who are developing a new generation of drugs against the avian influenza virus H5N1 by focusing their attention to this small portion of the chemical space.

Role of Secondary Sialic Acid Binding Sites in Influenza N1 Neuraminidase

Within influenza viral particles, the intricate balance between host cell binding and sialic acid receptor destruction is carefully maintained by the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, respectively. A major outstanding question in influenza biology is the function of a secondary sialic acid binding site on the NA enzyme. Through a series of Brownian dynamics (BD) simulations of the avian N1, human pandemic N2, and currently circulating pandemic (H1)N1 enzymes, we have probed the role of this secondary sialic acid binding site in the avian N1 subtype. Our results suggest that electrostatic interactions at the secondary and primary sites in avian NA may play a key role in the recognition process of the sialic acid receptors and catalytic efficiency of NA. This secondary site appears to facilitate the formation of complexes with the NA protein and the sialic acid receptors, as well as provide HA activity to a lesser extent. Moreover, this site is able to steer inhibitor binding as well, albeit with reduced capacity in N1, and may have potential implications for drug resistance or optimal inhibitor design. Although the secondary sialic acid binding site has previously been shown to be nonconserved in swine NA strains, our investigations of the currently circulating pandemic H1N1 strain of swine origin appears to have retained some of the key features of the secondary sialic acid binding site. Our results indicate possible lowered HA activity for this secondary sialic acid site, which may be an important event in the emergence of the current pandemic strain.

Design of novel, potent neuraminidase inhibitor for H5N1 avian influenza using molecular docking, multinuclear NMR and DSC methods

To probe more effective inhibitors for neuraminidase subtype N1, four potential inhibitors were synthetically designed by substitution at the C5 position of oseltamivir to provide additional interaction with the 150-cavity, a well-known active site in the neuraminidase subtype N1. Molecular docking with H5N1 and intermolecular interaction with model membrane to predict their drug effectiveness have been carried out. A comparison of four potential inhibitors with oseltamivir indicates that the glycyl derivative of oseltamivir has the most profound effects on the membrane, compared to the other derivatives. It seems to be the most promising derivative for further pharmacological evaluation as an neuraminidase inhibitor.

Molecular prediction of oseltamivir efficiency against probable influenza A (H1N1-2009) mutants: molecular modeling approach

To predict the susceptibility of the probable 2009 influenza A (H1N1-2009) mutant strains to oseltamivir, MD/LIE approach was applied to oseltamivir complexed with the most frequent drug-resistant strains of neuraminidase subtypes N1 and N2: two mutations on the framework residues (N294S and H274Y) and the two others on the direct-binding residues (E119V and R292K) of oseltamivir. Relative to those of the wild type (WT), loss of drug-target interaction energies, especially in terms of electrostatic contributions and hydrogen bonds were dominantly established in the E119V and R292K mutated systems. The inhibitory potencies of oseltamivir towards the WT and mutants were predicted according to the ordering of binding-free energies: WT (-12.3 kcal mol(-1)) > N294S (-10.4 kcal mol(-1)) > H274Y (-9.8 kcal mol(-1)) > E119 V (-9.3 kcal mol(-1)) > R292K (-7.7 kcal mol(-1)), suggesting that the H1N1-2009 influenza with R292K substitution, perhaps, conferred a high level of oseltamivir resistance, while the other mutants revealed moderate resistance levels. This result calls for an urgent need to develop new potent anti-influenza agents against the next pandemic of potentially higher oseltamivir-resistant H1N1-2009 influenza.

Infiltration of Water Molecules into the Oseltamivir-Binding Site of H274Y Neuraminidase Mutant Causes Resistance to Oseltamivir

The oseltamivir-resistant neuraminidase mutant, His274Tyr (H274Y), has recently been identified in humans. The objective of our present research is to elucidate the origin of resistance of the H274Y mutation to oseltamivir (OTV) at the molecular level via molecular dynamics simulation. For this purpose, the binding free energies of OTV with the wild-type N1 subtype and with the H274Y mutant were calculated using the linear interaction energy method. The nonbonded interaction energies between OTV and active-site residues were also calculated to investigate the contribution of nonbonded interactions to the total binding free energy. Our molecular dynamics trajectories revealed marked differences between these two structures, particularly in the binding modes of OTV. Contrary to the existing prediction, a salt bridge between Glu276 and Arg224 was retained in the OTV-bound H274Y mutant throughout the simulation. This study reveals that water molecules can infiltrate into the binding site of the H274Y mutant, suggesting that the origin of H274Y resistance to OTV is caused by penetration of water molecules into the binding site and not by disruption of the Glu276-Arg224 salt bridge. These results will be useful for the rational design of neuraminidase inhibitors having high potency against known drug-resistant H5N1 mutants.

Computational design of novel, high-affinity neuraminidase inhibitors for H5N1 avian influenza virus

To propose more effective inhibitors for neuraminidase subtype N1, four potential inhibitors were molecularly designed by substitution at the C3 position of oseltamivir to give additional interaction with the 150-cavity, since a new cavity known as the ‘150-cavity’ adjacent to the well-known active site has been found in the neuraminidase subtype N1. We calculated the binding free energy of both oseltamivir and the newly designed inhibitors for subtype N1, using molecular dynamics simulations, to predict their drug effectiveness. When the drug effectiveness of four potential inhibitors is compared with that of oseltamivir, we discovered a highly potent neuraminidase inhibitor, which exhibited much higher binding affinity to subtype N1 than oseltamivir (−17.77 vs. −8.06kcal/mol).

Wild Bird’s-eye View of Influenza Virus A(H1N1) Phylogenetic Evolution

Wild bird fecal samples collected and characterized by the USDA as part of a national surveillance effort were sequenced to study the genetic relatedness of avian, swine, and human H1 and N1 subtypes. Our results find that the 2009 H1N1 human outbreak is closely related to swine virus, but falls into different clades in the H1 and N1 trees. Further, there is evidence of multiple viral genetic exchanges between birds and swine. Ongoing research across host species contributes to an understanding of the circulation of influenza viruses.

Detection and Differentiation of Four Poultry Diseases Using Asymmetric Reverse Transcription Polymerase Chain Reaction in Combination with Oligonucleotide Microarrays

Asymmetric reverse transcription polymerase chain reaction (RT-PCR) and microarrays were combined to distinguish 4 viruses, including Avian influenza virus (AIV), Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), and Infectious bursal disease virus (IBDV), and hemagglutinin (HA) subtypes H5, H7, and H9, and neuraminidase (NA) subtypes N1 and N2 of AIV. The AIV matrix protein (M), and HA and NA genes, IBV nucleoprotein (NP) gene, NDV NP gene, and IBDV A fragment gene were cloned into plasmids. These genes were amplified from these positive recombinant plasmids, which included the inserted target genes by PCR. The PCR products were purified and printed on the amino-modified slides as the probes. RNA was extracted from samples and labeled by asymmetric RT-PCR using a cyanine (Cy)3-labeled primers. The labeled complementary (c)DNA was hybridized to the probes immobilized on the glass slides. After hybridization, the microarrays were scanned, and the hybridization pattern agreed perfectly with the known location of each probe. No cross-hybridization could be detected. Results demonstrated that microarray based on asymmetric RT-PCR is an effective way to distinguish AIV, IBV, NDV, and IBDV simultaneously.

The Special Neuraminidase Stalk-Motif Responsible for Increased Virulence and Pathogenesis of H5N1 Influenza A Virus

The variation of highly pathogenic avian influenza H5N1 virus results in gradually increased virulence in poultry, and human cases continue to accumulate. The neuraminidase (NA) stalk region of influenza virus varies considerably and may associate with its virulence. The NA stalk region of all N1 subtype influenza A viruses can be divided into six different stalk-motifs, H5N1/2004-like (NA-wt), WSN-like, H5N1/97-like, PR/8-like, H7N1/99-like and H5N1/96-like. The NA-wt is a special NA stalk-motif which was first observed in H5N1 influenza virus in 2000, with a 20-amino acid deletion in the 49th to 68th positions of the stalk region. Here we show that there is a gradual increase of the special NA stalk-motif in H5N1 isolates from 2000 to 2007, and notably, the special stalk-motif is observed in all 173 H5N1 human isolates from 2004 to 2007. The recombinant H5N1 virus with the special stalk-motif possesses the highest virulence and pathogenicity in chicken and mice, while the recombinant viruses with the other stalk-motifs display attenuated phenotype. This indicates that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The gradually increasing emergence of the special NA stalk-motif in H5N1 isolates, especially in human isolates, deserves attention by all.

Study on evolutionary origin of influenza A virus (H1N1) based on HA gene

To determine the evolutionary rate and divergence time of influenza A virus HA gene isolated recently worldwide pandemic and explore the origin and its transmission. A total of 344 H1 sequences available in the GenBank (including 248 isolated from human, 84 from swine, 11 from avian, and 1 from ferret) and 7 isolated in Shanghai were collected. The nucleotide substitution rate and time to most recent common ancestor (tMRCA) was calculated using molecular clock theory and Bayesian Skyline Plot (BSP) based on Markov chain Monte Carlo. Then genetic phylogeny was constructed referring to posterior distribution. It was found that H1 sequences in the US from human, swine and avian were clustered significantly with swine H1 ones from Asia phylogenetically (Cluster US). The second cluster (Cluster Eurasian Human) nearly consisted of human H1 sequences isolated in other regions. The third cluster (Cluster Eurasian Animal) consisted of swine and avian H1 sequences from China and Italy respectively. As for all the H1 sequences, the evolutionary rate was of 2.57 x 10(-3) substitutions/site per year averagely (95% Highest Posterior Density: 1.96 x 10(-3) - 3.03 x 10(-3)/site per year). The estimated dates for tMRCA of human H1 in Europe and swine H1 in the mainland of China were the earliest, with the corresponding rates of 6.46 x 10(-3)/site per year and 0.97 x 10(-3)/site per year respectively. The tMRCAs of human and swine H1 sequences from the US were similar, with the rates of 5.86 x 10(-3)/site per year and 5.02 x 10(-3)/site per year. The present flu outbreak was possibly induced by long-term circulation of influenza A virus (H1N1) in human population and swine herds in America. There was no evidence proving that influenza virus in China involved in the present outbreak.

ChemInform Abstract: Binding Interaction Analysis of the Active Site and Its Inhibitors for Neuraminidase (N1 Subtype) of Human Influenza Virus by the Integration of Molecular Docking, FMO Calculation and 3D‐QSAR CoMFA Modeling.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

CS-8958, a Prodrug of the New Neuraminidase Inhibitor R-125489, Shows Long-Acting Anti-Influenza Virus Activity

Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for the treatment of and prophylaxis against influenza. In this paper, the new potent NA inhibitor R-125489 is reported for the first time. R-125489 inhibited the NA activities of various type A and B influenza viruses, including subtypes N1 to N9 and oseltamivir-resistant viruses. The survival effect of R-125489 was shown to be similar to that of zanamivir when administered intranasally in a mouse influenza virus A/Puerto Rico/8/34 infection model. Moreover, we found that the esterified form of R-125489 showed improved efficacy compared to R-125489 and zanamivir, depending on the acyl chain length, and that 3-(O)-octanoyl R-125489 (CS-8958) was the best compound in terms of its life-prolonging effect (P < 0.0001, compared to zanamivir) in the same infection model. A prolonged survival effect was observed after a single administration of CS-8958, even if it was given 7 days before infection. It is suggested that intranasally administered CS-8958 works as a long-acting NA inhibitor and shows in vivo efficacy as a result of a single intranasal administration.

Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses

Accurate identification of hemagglutinin (HA) and neuraminidase (NA) subtypes of influenza A viruses is an integral part of monitoring programs targeting avian influenza viruses (AIV). Use of highly sensitive molecular screening methods such as pan influenza-specific real-time RT-PCR (rRT-PCR) yields an increasing number of samples which are positive for AIV RNA but negative by virus isolation and, therefore, require molecular, instead of serological, subtyping. We developed specific RT-PCR assays for all known nine AIV NA subtypes. Validation using 43 reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity. On basis of serial tenfold dilution series of reference isolates a benchmark value of C t 32 in an M gene-specific rRT-PCR became evident below which all nine NA subtypes were readily detectable by the subtype-specific RT-PCRs. For subtypes N1, N2, N4 and N6 detection was extended to dilutions with C t values of up to 35. Diagnostic applicability of the whole set of conventional NA-specific RT-PCRs was evaluated by analysis of 119 different diagnostic samples from wild birds which proved to be positive for AIV by M gene-specific rRT-PCR. Diagnostic sensitivity and specificity was confirmed by sequencing NA amplicons from 41 field isolates generated from this set and by NA inhibition assays. A universal molecular HA/NA subtyping algorithm for rRT-PCR positive avian influenza virus monitoring samples is proposed which may complement classical serological subtyping of influenza A virus isolates.

Pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza A viruses

Pyrosequencing has been successfully used to monitor resistance in influenza A viruses to the first class of anti-influenza drugs, M2 blockers (adamantanes). In contrast to M2 blockers, resistance to neuraminidase (NA) inhibitors (NAIs) is subtype- and drug-specific. Here, we designed a pyrosequencing assay for detection of the most commonly reported mutations associated with resistance to NAIs, a newer class of anti-influenza drugs. These common mutations occur at residues: H274 (N1), E119 (N2), R292 (N2), and N294 (N2) in seasonal influenza A viruses. Additionally, we designed primers to detect substitutions at D151 in NAs of N1 and N2 subtypes. This assay allows detection of mutations associated with resistance not only in grown viruses but also in clinical specimens, thus reducing the time needed for testing and providing an advantage for disease outbreak investigation and management. The pyrosequencing approach also allows the detection of mixed populations of virus variants at positions of interest. Analysis of viruses in the original clinical specimens reduces the potential for introducing genetic variance in the virus population due to selection by cell culture. Our results showed that, in at least one instance, a D151E change seen in N1NA after virus propagation in cell culture was not detected in the original clinical specimen. Although the pyrosequencing assay allows high throughput screening for established genetic markers of antiviral resistance, it is not a replacement for the NA inhibition assays due to insufficient knowledge of the molecular mechanisms of the NAI-resistance.

Binding Interaction Analysis of the Active Site and Its Inhibitors for Neuraminidase (N1 Subtype) of Human Influenza Virus by the Integration of Molecular Docking, FMO Calculation and 3D-QSAR CoMFA Modeling

Recently, the worldwide spread of A/H5N1 avian influenza with high virulence has highlighted the potential threat of human influenza pandemic. Tamiflu and Relenza are currently the only two anti-influenza drugs targeting the neuraminidase (NA) enzyme of human influenza virus. Reports of the emergence of drug resistance further make the development of new potent anti-influenza inhibitors a priority. The X-ray crystallographic study of A/H5N1 avian influenza NA subtypes (Russell, R. J. Nature 2006, 443, 45-49) has demonstrated that there exist two genetically distinct groups, group-1 (N1, N4, N5 and N8) and group-2 (N2, N3, N6, N7 and N9), whose conformations are substantially different. The detailed comparison of their active sites has established, heretofore, the most accurate and solid molecular basis of structure and mechanism for the development of new anti-influenza drugs. In the present study, a three-dimensional structure of N1 subtype of human influenza type A virus (N1hA) has been generated by homology modeling using the X-ray crystallographic structure of N1 subtype of avian influenza virus (N1aA) as the template. Binding interaction analysis between the active site and its inhibitors has been performed by combining ab initio fragment molecular orbital (FMO) calculations and three-dimensional quantitative structure-activity relationship with comparative molecular field analysis (3D-QSAR CoMFA) modeling. Integrated with docking-based 3D-QSAR CoMFA modeling, molecular surface property (electrostatic and steric) mapping and FMO pair interaction analysis, a set of new receptor-ligand binding models and bioaffinity predictive models for rational design and virtual screening of more potent inhibitors of N1hA are established. In addition, the flexibility of the loop-150 of N1hA and N1aA has been examined by a series of molecular dynamics simulations.

Origins of Resistance Conferred by the R292K Neuraminidase Mutation via Molecular Dynamics and Free Energy Calculations

Point mutations in the influenza virus enzyme neuraminidase (NA) have been reported that lead to dramatic loss of activity for known NA inhibitors including the FDA approved sialic acid mimics zanamivir and oseltamivir. A more complete understanding of the molecular basis for such resistance is a critical component toward development of improved next-generation drugs. In this study, we have used explicit solvent all-atom molecular dynamics simulations, free energy calculations (MM-GBSA), and residue-based decomposition to model binding of four ligands with NA from influenza virus subtype N9. The goal is to elucidate which structural and energetic properties change as a result of a mutation at position R292K. Computed binding free energies show strong correlation with experiment (r(2) = 0.76), and an examination of individual energy components reveal that changes in intermolecular Coulombic terms (ΔEcoul) best describe the variation in affinity with structure (r(2) = 0.93). H-bond populations also parallel the experimental ordering (r = -0.96, r(2) = 0.86) reinforcing the view that electrostatics modulate binding in this system. Notably, in every case, the simulation results correctly predict that loss of binding occurs as a result of the R292K mutation. Per-residue binding footprints reveal that changes in ΔΔEcoul for R292K-wildtype at position 292 parallel the change in experimental fold resistance energies (ΔΔGR292K-WT) with S03 < S00 < S02 < S01. The footprints also reveal that the most potent ligands have (1) less reliance on R292 for intrinsic affinity, (2) enhanced binding via residues E119, E227, and E277, and (3) flatter ΔEcoul and ΔH-bond profiles. Improved resistance for S03 appears to be a function of the ligand's larger guanidinium group which leads to an increased affinity for wildtype NA while at the same time a reduction in favorable interactions localized to R292. Overall, the computational results significantly enhance experimental observations through quantification of specific interactions which govern molecular recognition along the N9-ligand binding interface.