A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Reduced sialidase activity of influenza A(H3N2) neuraminidase associated with positively charged amino acid substitutions.

Neuraminidase (NA) inhibitors (NAI), oseltamivir and zanamivir, are the main antiviral medications for influenza and monitoring of susceptibility to these antivirals is routinely done by determining 50 % inhibitory concentrations (IC50) with MUNANA substrate. During 2010-2019, levels of A(H3N2) viruses presenting reduced NAI inhibition (RI) were low (~0.75 %) but varied year-on-year. The highest proportions of viruses showing RI were observed during the 2013-2014, 2016-2017 and 2017-2018 Northern Hemisphere seasons. The majority of RI viruses were found to contain positively charged NA amino acid substitutions of N329K, K/S329R, S331R or S334R, being notably higher during the 2016-2017 season. Sialidase activity kinetics were determined for viruses of RI phenotype and contemporary wild-type (WT) viruses showing close genetic relatedness and displaying normal inhibition (NI). RI phenotypes resulted from reduced sialidase activity compared to relevant WT viruses. Those containing S329R or N329K or S331R showed markedly higher Km for the substrate and Ki values for NAIs, while those with S334R showed smaller effects. Substitutions at N329 and S331 disrupt a glycosylation sequon (NDS), confirmed to be utilised by mass spectrometry. However, gain of positive charge at all three positions was the major factor influencing the kinetic effects, not loss of glycosylation. Because of the altered enzyme characteristics NAs carrying these substitutions cannot be assessed reliably for susceptibility to NAIs using standard MUNANA-based assays due to reductions in the affinity of the enzyme for its substrate and the concentration of the substrate usually used.

A natural variant in ANP32B impairs influenza virus replication in human cells.

Viruses require host factors to support their replication, and genetic variation in such factors can affect susceptibility to infectious disease. Influenza virus replication in human cells relies on ANP32 proteins, which are involved in assembly of replication-competent dimeric influenza virus polymerase (FluPol) complexes. Here, we investigate naturally occurring single nucleotide variants (SNV) in the human Anp32A and Anp32B genes. We note that variant rs182096718 in Anp32B is found at a higher frequency than other variants in either gene. This SNV results in a D130A substitution in ANP32B, which is less able to support FluPol activity than wild-type ANP32B and binds FluPol with lower affinity. Interestingly, ANP32B-D130A exerts a dominant negative effect over wild-type ANP32B and interferes with the functionally redundant paralogue ANP32A. FluPol activity and virus replication are attenuated in CRISPR-edited cells expressing wild-type ANP32A and mutant ANP32B-D130A. We propose a model in which the D130A mutation impairs FluPol dimer formation, thus resulting in compromised replication. We suggest that both homozygous and heterozygous carriers of rs182096718 may have some genetic protection against influenza viruses.

Limited Predictability of Amino Acid Substitutions in Seasonal Influenza Viruses.

Seasonal influenza viruses repeatedly infect humans in part because they rapidly change their antigenic properties and evade host immune responses, necessitating frequent updates of the vaccine composition. Accurate predictions of strains circulating in the future could therefore improve the vaccine match. Here, we studied the predictability of frequency dynamics and fixation of amino acid substitutions. Current frequency was the strongest predictor of eventual fixation, as expected in neutral evolution. Other properties, such as occurrence in previously characterized epitopes or high Local Branching Index (LBI) had little predictive power. Parallel evolution was found to be moderately predictive of fixation. Although the LBI had little power to predict frequency dynamics, it was still successful at picking strains representative of future populations. The latter is due to a tendency of the LBI to be high for consensus-like sequences that are closer to the future than the average sequence. Simulations of models of adapting populations, in contrast, show clear signals of predictability. This indicates that the evolution of influenza HA and NA, while driven by strong selection pressure to change, is poorly described by common models of directional selection such as traveling fitness waves.

Design, synthesis, and bioassay of 4-thiazolinone derivatives as influenza neuraminidase inhibitors.

A series of 4-thiazolinone derivatives (D1-D58) were designed and synthesized. All of the derivatives were evaluated in vitro for neuraminidase (NA) inhibitory activities against influenza virus A (H1N1), and the inhibitory activities of the five most potent compounds were further evaluated on NA from two different influenza viral subtypes (H3N2 and B), and then their in vitro anti-viral activities were evaluated using the cytopathic effect (CPE) reduction assay. The results showed that the majority of the target compounds exhibited moderate to good NA inhibitory activity. Compound D18 presented the most potent inhibitory activity with IC50 values of 13.06 µM against influenza H1N1 subtype. Among the selected compounds, D18 and D41 turned out to be the most potent inhibitors against influenza virus H3N2 subtype (IC50 = 15.00 µM and IC50 = 14.97 µM, respectively). D25 was the most potent compound against influenza B subtype (IC50 = 16.09 µM). In addition, D41 showed low toxicity and greater potency than reference compounds Oseltamivir and Amantadine against N1-H275Y variant in cellular assays. The structure-activity relationship (SAR) analysis showed that introducing 4-CO2H, 4-OH, 3-OCH3-4-OH substituted benzyl methylene can greatly improve the activity of 4-thiazolinones. Further SAR analysis indicated that 4-thiazolinone and ferulic acid fragments are necessary fragments of target compounds for inhibiting NA. Molecular docking was performed to study the interaction between compound D41 and the active site of NA. This study may providing important information for new drug development for anti-influenza virus including mutant influenza virus.

Development of A4 antibody for detection of neuraminidase I223R/H275Y-associated antiviral multidrug-resistant influenza virus.

The emergence and spread of antiviral drug-resistant viruses have been a worldwide challenge and a great concern for patient care. We report A4 antibody specifically recognizing and binding to the mutant I223R/H275Y neuraminidase and prove the applicability of A4 antibody for direct detection of antiviral multidrug-resistant viruses in various sensing platforms, including naked-eye detection, surface-enhanced Raman scattering-based immunoassay, and lateral flow system. The development of the A4 antibody enables fast, simple, and reliable point-of-care assays of antiviral multidrug-resistant influenza viruses. In addition to current influenza virus infection testing methods that do not provide information on the antiviral drug-resistance of the virus, diagnostic tests for antiviral multidrug-resistant viruses will improve clinical judgment in the treatment of influenza virus infections, avoid the unnecessary prescription of ineffective drugs, and improve current therapies.

Discovery of a non-zwitterionic oseltamivir analogue as a potent influenza a neuraminidase inhibitor.

The most of potent neuraminidase inhibitors as zwitterions with poor lipophilicity suffered from the poor oral bioavailability. Herein, we describe a rational journey to discover a non-zwitterionic neuraminidase inhibitor 24a containing urea. It showed potent inhibitions against neuraminidases from group 1(H5N1 and H1N1) and group 2 (H3N2) subtypes and exhibited more strong inhibitory activities against neuraminidases from H274Y mutants than oseltamivir carboxylate. Whether administrated by orally or intravenous injection, the pharmacokinetic profile of compound 24a in SD rats were improved compared to oseltamivir carboxylate.

Rapid detection of an I38T amino acid substitution in influenza polymerase acidic subunit associated with reduced susceptibility to baloxavir marboxil.

BACKGROUND: The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved in February 2018 for the treatment of influenza virus infection in Japan. In vitro studies have revealed that an I38T substitution in the polymerase acidic subunit (PA) is associated with reduced susceptibility of influenza viruses to baloxavir. OBJECTIVES: Development of a rapid and simple method for monitoring influenza A(H1N1)pdm09, A(H3N2), and B viruses possessing the I38T substitution in PA. METHODS: Three assays were developed based on RNase H2-dependent PCR (rhPCR) and named A/H1pdm PA_I38T rhPCR, A/H3 PA_I38T rhPCR, and B PA_I38T rhPCR. The assays were evaluated using cDNAs synthesized from in vitro-transcribed PA gene RNA controls, RNAs purified from viruses isolated in the 2017/2018 and 2018/2019 influenza seasons, and RNAs purified from clinical specimens collected in the 2018/2019 influenza season. RESULTS: The assays developed in this study accurately discriminated PA I38 and PA T38 with high sensitivity. CONCLUSIONS: Our assays should be considered a powerful tool for monitoring the emergence of baloxavir-resistant influenza viruses.

Impact of the Baloxavir-Resistant Polymerase Acid I38T Substitution on the Fitness of Contemporary Influenza A(H1N1)pdm09 and A(H3N2) Strains.

BACKGROUND: Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence. METHODS: Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR. RESULTS: I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments. CONCLUSION: The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.

Structures of influenza A virus RNA polymerase offer insight into viral genome replication.

Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPolA) composed of PB1, PB2 and PA subunits3-5. Although the high-resolution crystal structure of FluPolA of bat influenza A virus has previously been reported6, there are no complete structures available for human and avian FluPolA. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase7-10-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPolA from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPolA forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPolA bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPolA dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPolA dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPolA, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPolA that could be targeted in the development of antiviral drugs.

Effect of aloin on viral neuraminidase and hemagglutinin-specific T cell immunity in acute influenza.

BACKGROUND: Millions of people are infected by the influenza virus worldwide every year. Current selections of anti-influenza agents are limited and their effectiveness and drug resistance are still of concern. PURPOSE: Investigation on in vitro and in vivo effect of aloin from Aloe vera leaves against influenza virus infection. METHODS: In vitro antiviral property of aloin was measured by plaque reduction assay in which MDCK cells were infected with oseltamivir-sensitive A(H1N1)pdm09, oseltamivir-resistant A(H1N1)pdm09, H1N1 or H3N2 influenza A or with influenza B viruses in the presence of aloin. In vivo activity was tested in H1N1 influenza virus infected mice. Aloin-mediated inhibition of influenza neuraminidase activity was tested by MUNANA assay. Aloin treatment-mediated modulation of anti-influenza immunity was tested by the study of hemagglutinin-specific T cells in vivo. RESULTS: Aloin significantly reduced in vitro infection by all the tested strains of influenza viruses, including oseltamivir-resistant A(H1N1)pdm09 influenza viruses, with an average IC50 value 91.83 ± 18.97 µM. In H1N1 influenza virus infected mice, aloin treatment (intraperitoneal, once daily for 5 days) reduced virus load in the lungs and attenuated body weight loss and mortality. Adjuvant aloin treatment also improved the outcome with delayed oseltamivir treatment. Aloin inhibited viral neuraminidase and impeded neuraminidase-mediated TGF-ß activation. Viral neuraminidase mediated immune suppression with TGF-ß was constrained and influenza hemagglutinin-specific T cell immunity was increased. There was more infiltration of hemagglutinin-specific CD4+ and CD8+ T cells in the lungs and their production of effector cytokines IFN-γ and TNF-α was boosted. CONCLUSION: Aloin from Aloe vera leaves is a potent anti-influenza compound that inhibits viral neuraminidase activity, even of the oseltamivir-resistant influenza virus. With suppression of this virus machinery, aloin boosts host immunity with augmented hemagglutinin-specific T cell response to the infection. In addition, in the context of compromised benefit with delayed oseltamivir treatment, adjuvant aloin treatment ameliorates the disease and improves survival. Taken together, aloin has the potential to be further evaluated for clinical applications in human influenza.

The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A/Hong Kong/4801/2014 vaccine strain.

A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines.

Baloxavir marboxil susceptibility of influenza viruses from the Asia-Pacific, 2012-2018.

Baloxavir Marboxil (BXM) is an influenza polymerase inhibitor antiviral that binds to the endonuclease region in the PA subunit of influenza A and B viruses. To establish the baseline susceptibility of viruses circulating prior to licensure of BXM and to monitor for susceptibility post-BXM use, a cell culture-based focus reduction assay was developed to determine the susceptibility of 286 circulating seasonal influenza viruses, A(H1N1)pdm09, A(H3N2), B (Yamagata/Victoria) lineage viruses, including neuraminidase inhibitor (NAI) resistant viruses, to Baloxavir Acid (BXA), the active metabolic form of BXM. BXA was effective against all influenza subtypes tested with mean EC50 values (minimum-maximum) of 0.7 ±â€¯0.5 nM (0.1-2.1 nM), 1.2 ±â€¯0.6 nM (0.1-2.4), 7.2 ±â€¯3.5 nM (0.7-14.8), and 5.8 ±â€¯4.5 nM (1.8-15.5) obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses, respectively. Using reverse genetics, amino acid substitutions known to alter BXA susceptibility were introduced into the PA protein resulting in EC50 fold change increases that ranged from 2 to 65. Our study demonstrates that currently circulating viruses are susceptible to BXA and that the newly developed focus reduction assay is well suited to susceptibility monitoring in reference laboratories.

Antibody-free digital influenza virus counting based on neuraminidase activity.

There is large demand for a quantitative method for rapid and ultra-sensitive detection of the influenza virus. Here, we established a digital influenza virus counting (DIViC) method that can detect a single virion without antibody. In the assay, a virion is stochastically entrapped inside a femtoliter reactor array device for the fluorogenic assay of neuraminidase, and incubated for minutes. By analyzing 600,000 reactors, the practical limit of detection reached the order of 103 (PFU)/mL, only 10-times less sensitive than RT-PCR and more than 1000-times sensitive than commercial rapid test kits (RIDTs). Interestingly, neuraminidase activity differed among virions. The coefficient of variance was 30-40%, evidently broader than that of alkaline phosphatase measured as a model enzyme for comparison, suggesting the heterogeneity in size and integrity among influenza virus particles. Sensitivity to oseltamivir also differed between virions. We also tested DIViC using clinical gargle samples that imposes less burden for sampling while with less virus titre. The comparison with RIDTs showed that DIViC was largely superior to RIDTs in the sensitivity with the clinical samples although a few false-positive signals were observed in some clinical samples that remains as a technical challenge.

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.

Genome-wide characterization of the seasonal H3N2 virus in Shanghai reveals natural temperature-sensitive strains conferred by the I668V mutation in the PA subunit.

Seasonal H3N2 influenza viruses are recognized as major epidemic viruses, exhibiting complex seasonal patterns in regions with temperate climates. To investigate the influence of viral evolution and mutations on the seasonality of influenza, we performed a genome-wide analysis of samples collected from 62 influenza A/H3N2-infected patients in Shanghai during 2016-2017. Phylogenetic analysis of all eight segments of the influenza A virus revealed that there were two epidemic influenza virus strains circulating in the 2016-2017 winter season (2016-2017win) and 2017 summer season (2017sum). Replication of the two epidemic viral strains at different temperatures (33, 35, 37, and 39 °C) was measured, and the correlation of the mutations in the two epidemic viral strains with temperature sensitivity and viral replication was analyzed. Analysis of the replication kinetics showed that replication of the 2016-2017win strains was significantly restricted at 39 °C compared with that of the 2017sum strains. A polymerase activity assay and mutational analysis demonstrated that the PA I668V mutation of the 2016-2017win viruses suppressed polymerase activity in vitro at high temperatures. Taken together, these data suggest that the I668V mutation in the PA subunit of the 2016-2017win strains may confer temperature sensitivity and attenuate viral replication and polymerase activity; meanwhile, the 2017sum strains maintained virulence at high temperatures. These findings highlight the importance of certain mutations in viral adaptation and persistence in subsequent seasons.

Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies.

Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.

Neuraminidase inhibitor susceptibility and neuraminidase enzyme kinetics of human influenza A and B viruses circulating in Thailand in 2010-2015.

Amino acid substitutions within or near the active site of the viral neuraminidase (NA) may affect influenza virus fitness. In influenza A(H3N2) and B viruses circulating in Thailand between 2010 and 2015, we identified several NA substitutions that were previously reported to be associated with reduced inhibition by NA inhibitors (NAIs). To study the effect of these substitutions on the enzymatic properties of NA and on virus characteristics, we generated recombinant influenza viruses possessing either a wild type (WT) NA or an NA with a single I222V, S331G, or S331R substitution [in influenza A(H3N2) viruses] or a single D342S, A395T, A395V, or A395D NA substitution (in influenza B viruses). We generated recombinant (7:1) influenza A and B viruses on the genetic background of A/Puerto Rico/8/1934 (A/PR/8, H1N1) or B/Yamanashi/166/1998 (B/YAM) viruses, respectively. In contrast to the expected phenotypes, all the recombinant influenza A(H3N2) and B viruses carrying putative NA resistance substitutions were susceptible to NAIs. The Km and Vmax for the NAs of A/PR8-S331G and A/PR8-S331R viruses were higher than for the NA of WT virus, and the corresponding values for the B/YAM-D342S virus were lower than for the NA of WT virus. Although there was initial variation in the kinetics of influenza A and B viruses' replication in MDCK cells, their titers were comparable to each other and to WT viruses at later time points. All introduced substitutions were stable except for B/YAM-D342S and B/YAM-A395V which reverted to WT sequences after three passages. Our data suggest that inferring susceptibility to NAIs based on sequence information alone should be cautioned. The impact of NA substitution on NAI resistance, viral growth, and enzymatic properties is viral context dependent and should be empirically determined.

The hydrophobic side chain of oseltamivir influences type A subtype selectivity of neuraminidase inhibitors.

Neuraminidase, which plays a critical role in the influenza virus life cycle, is a target for new therapeutic agents. The study of structure-activity relationships revealed that the C-5 position amino group of oseltamivir was pointed to 150-cavity of the neuraminidase in group 1. This cavity is important for selectivity of inhibitors against N1 versus N2 NA. A serial of influenza neuraminidase inhibitors with the oseltamivir scaffold containing lipophilic side chains at the C-5 position have been synthesized and evaluated for their influenza neuraminidase inhibitory activity and selectivity. The results indicated that compound 13o (H5N1 IC50  = 0.1 ± 0.04 µm, H3N2 IC50  = 0.26 ± 0.18 µm) showed better inhibitory activity and selectivity against the group 1 neuraminidase. This study may provide a clue to design of better group 1 neuraminidase inhibitors.

Characterization of the neuraminidase genes from human influenza A viruses circulating in Iran from 2010 to 2015.

BACKGROUND: Characterization of influenza viruses is critical for detection of new emerging variants. Herein, we analyzed the genetic diversity and drug susceptibility of the neuraminidase gene (NAs) expressed by influenza A/H1N1pdm09 and A/H3N2 viruses circulating in Iran from 2010 to 2015. METHODS: We genetically analyzed the NAs of 38 influenza A/H1N1pdm09 and 35 A/H3N2 isolates. RESULTS: The Iranian A/H1N1pdm09 viruses belonged to seven genogroups/subgenogroups, with the dominant groups being genogroups 6B and 6C. The A/H3N2 isolates fell into six gneogroups/subgenogroups, with the dominant genogroups being 3C and 3C.2a. The most common mutations detected among the A/H1N1pdm09 viruses included N44S, V106I, N200S, and N248D. All H1N1pdm09 viruses were genetically susceptible to the NAIs. However, one A/H1N1pdm09 virus from the 2013-2014 season possessed an NA-S247N mutation, which reduces the susceptibility to oseltamivir. In case of H3N2, none of the analyzed Iranian strains carried a substitution that might affect its susceptibility to NAIs. CONCLUSION: The ongoing evolution of influenza viruses and the detect of influenza viruses with reduced susceptibility to NAIs warrants continuous monitoring of the circulating strains.