Neuraminidase Inhibitor Drugs Research Articles

Virtual Screening of Indonesian Herbal Compounds with Neuraminidase Inhibitor Activity against N2 Influenza Virus Protein: An in silico Study

Background: Neuraminidase inhibitor (NAI) is one of anti-influenza drugs recommended for use by the World Health Organization (WHO). However, after treatment with NAI drugs in human, resistance to influenza antiviral drugs is begun to rise. Therefore, identification of compounds from Indonesian herbal plants as natural inhibitors of the influenza virus neuraminidase protein needs to be conducted for the development of new anti-influenza drugs.Materials and methods: The crystal structure of the neuraminidase protein complex used in this study was obtained from the Protein Data Bank (PDB). Structure-based pharmacophore modeling was performed using LigandScout version 4.4.5 software. Indonesian herbal plant compounds were collected from the HerbalDB database. Protein and ligand processing was carried out using Autodock 4.2 software. The 3D interaction visualization was carried out with Autodock software, while 2D interaction visualization was carried out with LigPlot software. To determine the toxicity and drug-likeliness of the ligand, the test ligands that had the best docking results were predicted using SwissADME and AdmetSAR.Results: From the virtual screening results, 24 hits were found, and five compounds had the best binding energy among the 24 tested compounds, these were pollenitin (ΔG=-7.22 kcal/mol), OPC-4:0 (ΔG=-7.11 kcal/mol), 6-hydroxykaempferol (ΔG=-7.08 kcal/mol), 5,8-dihydroxy-7,4'-dimethoxyflavone (ΔG=-7.07 kcal/mol), and 3,5,6,7-tetrahydroxy-4'-methoxyflavone (ΔG=-6.95 kcal/mol). The best five compounds were then chosen for further analysis.Conclusion: OPC-4:0 is found to be the best compound for the NAI based on its binding energy, pharmacokinetics, toxicity, and drug-likeliness. Thus, OPC-4:0 might be a potential candidate as a NAI of HxN2 virus. Keywords: influenza, molecular docking, neuraminidase, resistance, virtual screening

Virtual Screening of Indonesian Herbal Compounds with Neuraminidase Inhibitor Activity against N2 Influenza Virus Protein: An in silico Study

Background: Neuraminidase inhibitor (NAI) is one of anti-influenza drugs recommended for use by the World Health Organization (WHO). However, after treatment with NAI drugs in human, resistance to influenza antiviral drugs is begun to rise. Therefore, identification of compounds from Indonesian herbal plants as natural inhibitors of the influenza virus neuraminidase protein needs to be conducted for the development of new anti-influenza drugs.Materials and methods: The crystal structure of the neuraminidase protein complex used in this study was obtained from the Protein Data Bank (PDB). Structure-based pharmacophore modeling was performed using LigandScout version 4.4.5 software. Indonesian herbal plant compounds were collected from the HerbalDB database. Protein and ligand processing was carried out using Autodock 4.2 software. The 3D interaction visualization was carried out with Autodock software, while 2D interaction visualization was carried out with LigPlot software. To determine the toxicity and drug-likeliness of the ligand, the test ligands that had the best docking results were predicted using SwissADME and AdmetSAR.Results: From the virtual screening results, 24 hits were found, and five compounds had the best binding energy among the 24 tested compounds, these were pollenitin (ΔG=-7.22 kcal/mol), OPC-4:0 (ΔG=-7.11 kcal/mol), 6-hydroxykaempferol (ΔG=-7.08 kcal/mol), 5,8-dihydroxy-7,4'-dimethoxyflavone (ΔG=-7.07 kcal/mol), and 3,5,6,7-tetrahydroxy-4'-methoxyflavone (ΔG=-6.95 kcal/mol). The best five compounds were then chosen for further analysis.Conclusion: OPC-4:0 is found to be the best compound for the NAI based on its binding energy, pharmacokinetics, toxicity, and drug-likeliness. Thus, OPC-4:0 might be a potential candidate as a NAI of HxN2 virus. Keywords: influenza, molecular docking, neuraminidase, resistance, virtual screening

Berberine as a new neuraminidase inhibitor drug: A systematic review

Influenza neuraminidase (NA) plays a main role in the viral replication of the influenza virus. It has been considered one of the targets for anti-influenza drugs. Anti-influenza drugs such as zanamivir, oseltamivir, and peramivir can fight the virus via the inhibition of NA. However, due to adverse reactions, the resistance of the viral strains and sudden changes in NA inhibitors, the identification of novel inhibitors is needed. Nature products such as berberine have been reported against influenza. In this systematic review, we have focused on the anti-influenza effects of berberine and its main role in the inhibition of NA of the virus. For this aim, "Berberine" was searched with "Influenza" or "flu" or "common cold" or "neuraminidase" in Web of Science, PubMed, Scopus, and Google Scholar databases from 1990 to April 2023. Studies have demonstrated that berberine and its derivatives have a wide range of biological effects such as antiviral effects against viruses like herpes simplex virus, human cytomegalovirus, and influenza A virus. The present study indicates that berberine and some of its derivatives are able to inhibit the influenza virus through NA blockade. Berberine is able to superimpose into the allosteric binding site and shows reversible non-competitive behavior in the ligand-receptor interaction for the inhibition of NA.

The global epidemic trend analysis of influenza type B drug resistance sites from 2006 to 2018.

Influenza is a severe respiratory viral infection that causes significant morbidity and mortality, due to annual epidemics and unpredictable pandemics. With the extensive use of neuraminidase inhibitor (NAI) drugs, the influenza B virus has carried different drug-resistant mutations. Thus, this study aimed to analyze the prevalence of drug-resistant mutations of the influenza B virus. Near full-length sequences of the neuraminidase (NA) region of all influenza B viruses from January 1, 2006, to December 31, 2018, were downloaded from public databases GISAID and NCBI. Multiple sequence alignments were performed using Clustal Omega 1.2.4 software. Subsequently, phylogenetic trees were constructed by FastTree 2.1.11 and clustered by ClusterPickergui_1.2.3.JAR. Then, the major drug resistance sites and surrounding auxiliary sites were analyzed by Mega-X and Weblogo tools. Among the amino acid sequences of NA from 2006 to 2018, only Clust04 in 2018 carried a D197N mutation of the NA active site, while other drug resistance sites were conserved without mutation. According to the Weblogo analysis, a large number of N198, S295, K373, and K375 mutations were found in the amino acid residues at the auxiliary sites surrounding D197, N294, and R374. We found the D197N mutation in Clust04 of the 2018 influenza B virus, with a large number of N198, S295, K373, and K375 mutations in the helper sites around N197, N294, and R374 from 2006 to 2018. NA inhibitors are currently the only kind of specific antiviral agent for the influenza B virus, although these mutations cause mild NAIs resistance.

Discovery of Multi‐Targets Neuraminidase Inhibitor Lead Compound Against Influenza H1N1 Virus A/WSN/33 Based on QSAR, Docking, Dynamics Simulation and Network Pharmacology

AbstractIn this paper, three compounds are discovered as lead compounds of multi‐targets neuraminidase inhibitor drugs against influenza H1N1 virus A/WSN/33 using QSAR, molecular docking, dynamics simulation and network pharmacology. Moreover, NEU1, PLA2G1B and STAT1 are especially related to therapeutic targets of influenza virus. Therefore, the present study can be helpful in the process of the rational drug design of anti‐influenza virus drugs and improve reliability of findings novel multi‐target drugs which may avoid drug‐resistance.

Baloxavir treatment of ferrets infected with influenza A(H1N1)pdm09 virus reduces onward transmission.

Influenza viruses cause seasonal outbreaks and pose a continuous pandemic threat. Although vaccines are available for influenza control, their efficacy varies each season and a vaccine for a novel pandemic virus manufactured using current technology will not be available fast enough to mitigate the effect of the first pandemic wave. Antivirals can be effective against many different influenza viruses but have not thus far been used extensively for outbreak control. Baloxavir, a recently licensed antiviral drug that targets the influenza virus endonuclease, has been shown to reduce virus shedding more effectively than oseltamivir, a widely used neuraminidase inhibitor drug. Thus it is possible that treatment with baloxavir might also interrupt onward virus transmission. To test this, we utilized the ferret model, which is the most commonly used animal model to study influenza virus transmission. We established a subcutaneous baloxavir administration method in ferrets which achieved similar pharmacokinetics to the approved human oral dose. Transmission studies were then conducted in two different locations with different experimental setups to compare the onward transmission of A(H1N1)pdm09 virus from infected ferrets treated with baloxavir, oseltamivir or placebo to naïve sentinel ferrets exposed either indirectly in adjacent cages or directly by co-housing. We found that baloxavir treatment reduced infectious viral shedding in the upper respiratory tract of ferrets compared to placebo, and reduced the frequency of transmission amongst sentinels in both experimental setups, even when treatment was delayed until 2 days post-infection. In contrast, oseltamivir treatment did not substantially affect viral shedding or transmission compared to placebo. We did not detect the emergence of baloxavir-resistant variants in treated animals or in untreated sentinels. Our results support the concept that antivirals which decrease viral shedding could also reduce influenza transmission in the community.

The Organization and Clustering of Giardial Lipid Raft Domains After Treatment with Oseltamivir by Direct Stochastic Optical Resolution Microscopy

Lipid rafts contain combinations of glycosphigolipids, cholesterol, and protein receptors organized into lipid microdomains that form ordered structures. They play a role in signal transduction in order for cells to respond efficiently to changes in their environment and have the ability to undergo self-assembly and disassembly. Proteomic analysis reveals the presence of several proteins with biological functions in giardial lipid rafts (gLRs), including variable surface proteins, kinases, and glycosylphosphatidylinositol anchored proteins, giving rise to the idea that these lipid rafts could be a potential drug target. Previous data has shown that oseltamivir (Tamiflu), a neuraminidase inhibitor and FDA-approved drug, disrupts the growth and differentiation of Giardia by inhibiting formation of gLRs. Recalcitrant giardiasis is frequently contributed to drug resistance with treatment failures to the commonly prescribed drugs, metronidazole or albendazole, providing a need for new drug targets. We have determined the nanoscale clustering characteristics of GM1 ganglioside, a functionally important glycolipid in lipid rafts, using dSTORM, before and after treatment with oseltamivir. GM1 was labeled with CTxB-AF647 and Giardia were fixed in 4% agarose to prevent motion during acquisition for optimal resolution for dSTORM. In untreated Giardia, we observe clusters of GM1 with a mean radius of 19.3nm, with single probe density at 0.75E-3nm−2 and multi-probe density at 3.5E-5nm−2. After treatment with oseltamivir, we see a significant decrease in the size of GM1 clusters, with a mean radius of 15.1nm. Single probe density significantly increased to 1.5E-3nm−2, and multi-probe density significantly decreased to 1.25E-5nm−2. These results validate that at the nanoscale, oseltamivir disrupts gLR assembly and could be used as an anti-giardial drug, targeting specific raft clusters.

Source of oseltamivir resistance due to single E119D and double E119D/H274Y mutations in pdm09H1N1 influenza neuraminidase.

Influenza epidemics are responsible for an average of 3-5 millions of severe cases and up to 500,000 deaths around the world. One of flu pandemic types is influenza A(H1N1)pdm09 virus (pdm09H1N1). Oseltamivir is the antiviral drug used to treat influenza targeting at neuraminidase (NA) located on the viral surface. Influenza virus undergoes high mutation rates and leads to drug resistance, and thus the development of more efficient drugs is required. In the present study, all-atom molecular dynamics simulations were applied to understand the oseltamivir resistance caused by the single E119D and double E119D/H274Y mutations on NA. The obtained results in terms of binding free energy and intermolecular interactions in the ligand-protein interface showed that the oseltamivir could not be well accommodated in the binding pocket of both NA mutants and the 150-loop moves out from oseltamivir as an "open" state. A greater number of water molecules accessible to the binding pocket could disrupt the oseltamivir binding with NA target as seen be high mobility of oseltamivir at the active site. Additionally, our finding could guide to the design and development of novel NA inhibitor drugs.

Severe cases of seasonal influenza in Russia in 2017-2018

Aim. Evaluation of herd immunity prior to the 2017-2018 influenza season, and characterization of influenza viruses isolated from severe or fatal influenza cases and from influenza cases in people vaccinated in the fall of 2017. Materials and methods. Evaluation of herd immunity in hemagglutination inhibition assay. Isolation of influenza viruses. Antigenic and genetic analysis. Results. Prior to epidemic season 33-47% of blood sera samples collected on the territory of Russia showed presence of protective antibody titers against vaccine strains of influenza A, 24-30% of samples — against B/Victoria. During 2017-2018 epidemic season 87 influenza A and B viruses were isolated. A(H1N1)pdm09 strains belonged to clade 6B.1, B/Yamagata strains to clade 3, and B/Victoria strains to clade 1A; they were antigenically similar to corresponding vaccine strains. A(H3N2) viruses belonged to clade 3C.2a and were difficult to characterize antigenically. One strain of influenza virus А(H1N1pdm09) was resistant to oseltamivir and had H275Y amino acid substitution in neuraminidase. All other isolates were susceptible to neuraminidase inhibitors. Conclusion. Influenza vaccination with vaccine effective against current circulating strains and treatment with neuraminidase inhibitor drugs at first manifestation of clinical signs of influenza disease are effective means of population protection against influenza.

Untargeted metabolomics analysis of the upper respiratory tract of ferrets following influenza A virus infection and oseltamivir treatment.

Influenza is a highly contagious respiratory disease that causes high global morbidity and mortality each year. The dynamics of an influenza infection on the host metabolism, and how metabolism is altered in response to neuraminidase inhibitor drug therapy, is still in its infancy but of great importance. We aim to investigate the suitability of ferret nasal wash samples for metabolomics-based analysis and characterization of influenza infections and oseltamivir treatment. Virological and metabolic analyses were performed on nasal wash samples collected from ferrets treated with oseltamivir or a placebo. Untargeted metabolomics was performed using a gas chromatography coupled withmass spectrometery(GC-MS) based protocol that comprised a retention time (RT) locked method and the use of a commercial metabolomics library. Ferret activity was reduced at 2-3 days post infection, which coincided with the highest influenza viral titre. The metabolomics data indicated a shift in metabolism during various stages of infection. The neuraminidase inhibitor oseltamivir created considerable downregulation of energy center metabolites (glucose, sucrose, glycine and glutamine), which generated high levels of branched amino acids. This further increased branched amino acid degradation and deregulation via glycerate-type intermediates and biosynthesis of fatty acids in oseltamivir-treated animals where abrogated weight loss was observed. Metabolomics was used to profile influenza infection and antiviral drug treatment in ferrets. This has the potential to provide indicators for the early diagnosis of influenza infection and assess the effectiveness of drug therapies.

Molecular characteristic analysis of neuraminidase genes of avian influenza virus H9N2 in environments in Weining, Guizhou Province during 2015-2017

Objective To understand the genetic variations of neuraminidase (NA) genes of avian influenza virus H9N2 in Weining, Guizhou Province, and to provide the scientific evidence for the prevention and control of avian influenza virus. Methods Ribonucleic acids (RNA) were extracted and NA genes were amplified and sequenced from 13 randomly selected H9N2 positive samples from the live poultry market (LPM) environments in north of Weining Yi and Hui and Miao autonomous county (Weining), Guizhou Province during 2015 to 2017. Then the homology, genetic evolution, and sites of stalk deletion areas, potential N-glycosylation, receptor binding regions and drug resistance of H9N2 subtype avian influenza viruses were analyzed by a series of bioinformation software. Results Homology analysis revealed that there were 93.0%-100.0% and 92.1%-100.0% similarity among 13 strains H9N2 avian influenza viruses in nucleotide and amino acid of the NA gene, respectively. All strains belonged to DK/HK/Y280/97 sub-lineage, but their genetic sources were complex and diverse. Thirteen strains had a stalk deletion of 3 amino acid residues TEI at positions 63-65 and 3 isolates had mutation QN to QK at positions 39-40. The potential N-glycosylation sites at amino acid residues 86, 146, 200, and 234 of the NA protein of all strains were highly conserved, while other N-glycosylation sites had quantity and site mutations. There were different mutation types at the three sialic acid binding site areas, especially at 399-404 area. All NA protease activity sites and key sites of the 13 strains had no mutations associated with resistance to the neuraminidase inhibitor drugs. Conclusions All 13 strains H9N2 viruses belongs to DK/HK/Y280/97 sub-lineage in Weining, Guizhou Province during 2015-2017, and their genetic sources are complex and diverse. The mutations on sites of stalk areas, potential N-glycosylation and sialic acid binding site areas are presented at different degrees. Hence, enhancing surveillance and controlling H9N2 avian influenza virus is necessary. Key words: Influenza A virus, H9N2 subtype; Neuraminidase gene; Molecular characteristic

Influenza B viruses circulated during last 5 years in Mongolia.

Influenza B virus-caused illness has recently been considered as an urgent public health problem due to substantial morbidity, mortality and life-threatening medical complications. In this study, we have reported the main characteristics of influenza B virus in Mongolia, including prevalence, lineages, suitability with vaccine strains and drug susceptibility against the virus. 15768 specimens were tested by qPCR for detecting influenza viruses. From positive specimens for influenza B virus, the clinical isolates were isolated using MDCK cells. Sequencing analysis, hemagglutination inhibition assay and Neuraminidase inhibitor (NAI) drug susceptibility testing were performed for the clinical isolates. Influenza B virus was around in 3.46% of the samples in Mongolia, and B/Victoria clade-1A and B/Yamagata clade-3 lineages were predominant. Importantly, it was confirmed that the lineages corresponded to the vaccine strains. Moreover, drug susceptibility tests revealed that some Mongolian clinical isolates showed reduced susceptibility to antiviral agents. Interestingly, G104R was identified as a novel mutation, which might have a significant role in drug resistance of the virus. These results describe the characteristics of influenza B viruses that have caused respiratory illness in the population of Mongolia between 2013 and 2017.

Fluorescent Neuraminidase Assay Based on Supramolecular Dye Capture After Enzymatic Cleavage.

A conceptually new type of enzymatic cleavage assay is reported that utilizes in situ supramolecular capture of the fluorescent product. A squaraine-derived substrate with large blocking groups at each end of its structure cannot be threaded by a tetralactam macrocycle until the blocking groups are removed by enzyme cleavage. A prototype design responds to viral neuraminidase, an indicator of influenza infection, and also measures susceptibility of the sample to neuraminidase inhibitor drugs. The substrate structure incorporates three key features: (a) a bis(4-amino-3-hydroxyphenyl)squaraine core with bright deep-red fluorescence and excellent photostability, (b) an N-methyl group at each end of the squaraine core that ensures fast macrocycle threading kinetics, and (c) sialic acid blocking groups that prevent macrocycle threading until they are removed by viral neuraminidase. The enzyme assay can be conducted in aqueous solution where dramatic colorimetric and fluorescence changes are easily observed by the naked eye. Alternatively, affinity capture beads coated with macrocycle can be used to immobilize the liberated squaraine and enable a range of heterogeneous analysis options. With further optimization, this new type of neuraminidase assay may be useful in a point of care clinic to rapidly diagnose influenza infection and also determine which of the approved antiviral inhibitor drugs is likely to be the most effective treatment for an individual patient. The assay design is generalizable and can be readily modified to monitor virtually any type of enzyme-catalyzed cleavage reaction.

The in vivo efficacy of neuraminidase inhibitors cannot be determined from the decay rates of influenza viral titers observed in treated patients

Antiviral therapy is a first line of defence against new influenza strains. Current pandemic preparations involve stock- piling oseltamivir, an oral neuraminidase inhibitor (NAI), so rapidly determining the effectiveness of NAIs against new viral strains is vital for deciding how to use the stockpile. Previous studies have shown that it is possible to extract the drug efficacy of antivirals from the viral decay rate of chronic infections. In the present work, we use a nonlinear mathematical model representing the course of an influenza infection to explore the possibility of extracting NAI drug efficacy using only the observed viral titer decay rates seen in patients. We first show that the effect of a time-varying antiviral concentration can be accurately approximated by a constant efficacy. We derive a relationship relating the true treatment dose and time elapsed between doses to the constant drug dose required to approximate the time- varying dose. Unfortunately, even with the simplification of a constant drug efficacy, we show that the viral decay rate depends not just on drug efficacy, but also on several viral infection parameters, such as infection and production rate, so that it is not possible to extract drug efficacy from viral decay rate alone.

Efficacy of delayed treatment of China-made Peramivir with repeated intravenous injections in a mouse influenza model: from clinical experience to basal experiment.

BackgroundChina-made Peramivir, an anti-influenza neuraminidase inhibitor drug, is manufactured and widely used in China. Although effective if initiated within 48 h of the onset of symptoms, yet we observed that this drug shows an inconclusive efficacy if treatment is delayed in clinical. Thus we evaluated the efficacy of delayed treatment of China-made Peramivir in a mouse model.MethodsThe mouse model of influenza infection was made and Peramivir was administered intravenously for 5 days following infection, and weight loss, lung index, viral shedding and survival rates were monitored.ResultsPeramivir (60 mg/kg · d, repeated intravenous injections, quaque die (QD) × 5 days) enhanced survival rate and suppressed weight loss when treatment was initiated 24, 36, 48, or even 60 h post-infection (p.i.) (p < 0.01), compared with the virus-untreated group, and efficacy was abolished at 72 h p.i.. However the efficacy of delayed treatment was dose dependent, with the highest dose (90 mg/kg · d) even showing efficacy at 72 h p.i.. Furthermore, Peramivir (60 mg/kg · d, repeated intravenous injections, QD × 5 days) also reduced the lung virus titer 24 and 36 h p.i. on day 5, and even at 48 and 60 h p.i. on day 7 after infection, and the lung index was also improved. What is interesting that the concentration of the drug was maintained in blood after infected.ConclusionsDelayed treatment with China-made Peramivir can reduce the severity of influenza disease, accelerate viral clearance and enhance the survival rate. This drug therefore shows good efficacy and is a promising candidate to control the influenza epidemic in China.

6SLN-lipo PGA specifically catches (coats) human influenza virus and synergizes neuraminidase-targeting drugs for human influenza therapeutic potential.

The purpose of this study was to develop a new compound to overcome influenza epidemics and pandemics as well as drug resistance. We synthesized a new compound carrying: (i) Neu5Acα2-6Galβ1-4GlcNAc (6SLN) for targeting immutable haemagglutinins (HAs) unless switched from human-type receptor preference; (ii) an acyl chain (lipo) for locking the compound with the viral HA via hydrophobic interactions; and (iii) a flexible poly-α-L-glutamic acid (PGA) for enhancing the compound solubility and for coating the viral surface, precluding accessibility of the PGA-coated virus to the negatively charged sialic acid on the host cell surface. 6SLN-lipo PGA appears to subvert binding of pandemic H1 and seasonal H3 HAs to receptors, as assessed by using guinea pig erythrocytes, which is critical for virus entry into host cells for multiplication. It shows high potency with IC50 values in the range of 300-500 nM against multiplication of both influenza pandemic H1N1/2009 and seasonal H3N2/2004 viruses in cell culture. It acts in synergism with either of the two FDA-approved neuraminidase inhibitor (NAI) clinical drugs, zanamivir (Relenza(®)) and oseltamivir carboxylate (active form of Tamiflu(®)), and it has the potential to aid NAI drugs to achieve complete clearance of the virus from the culture. 6SLN-lipo PGA is a new potential candidate drug for influenza control and is an attractive candidate for use in combination with an NAI drug for minimized toxicity, delayed development of resistance, prevention and treatment with the potential for eradication of human influenza.

Developments in the treatment of severe influenza

Cases of severe influenza may occur during seasonal epidemics, following sporadic zoonotic influenza A transmission from animal reservoirs or on a massive scale with the unpredictable emergence of a new pandemic influenza strain. Clinical experience identifies unmet medical need for additional therapies for influenza, in particular to treat severely unwell adults and children. During and following the pandemic of 2009, a wealth of data from hospitalized cases of influenza from many different countries accumulated and are now starting to emerge. Observational clinical data provide information about the efficacy of existing antiviral drugs in severely ill patients. The development pipeline for new therapies contains several promising agents which are focussed on a range of viral targets, and opens the possibility of combination antiviral therapy for the first time, which may be especially useful in clinically challenging cases. Advances in immunological methods and recombinant protein engineering support the potential for use of immunomodulating therapies as adjuncts in treatment of severe influenza. The main themes are the importance of treating severe influenza early, considering multiple therapy options and the relevance of observational clinical data to treatment of severely ill and risk groups. Clinicians, who may have only seen the media headlines following discussion of reviews which deal with randomized controlled trials of neuraminidase inhibitor drug use in mild uncomplicated influenza in the community, may be hesitant to prescribe these drugs. Observational data arising from treatment of severely ill individuals support use of these drugs early in illness and show improvement in outcomes associated with drug use.

Neuraminidase Inhibitors for Critically Ill Children With Influenza

Timely treatment with neuraminidase inhibitor (NAI) drugs appears to improve survival in adults hospitalized with influenza. We analyzed California surveillance data to determine whether NAI treatment improves survival in critically ill children with influenza. We analyzed data abstracted from medical records to characterize the outcomes of patients aged 0 to 17 years hospitalized in ICUs with laboratory-confirmed influenza from April 3, 2009, through September 30, 2012. Seven hundred eighty-four influenza cases aged <18 years hospitalized in ICUs had information on treatment. Ninety percent (532 of 591) of cases during the 2009 H1N1 pandemic (April 3, 2009-August 31, 2010) received NAI treatment compared with 63% (121 of 193) of cases in the postpandemic period (September 1, 2010-September 30, 2012; P < .0001). Of 653 cases NAI-treated, 38 (6%) died compared with 11 (8%) of 131 untreated cases (odds ratio = 0.67, 95% confidence interval: 0.34-1.36). In a multivariate model that included receipt of mechanical ventilation and other factors associated with disease severity, the estimated risk of death was reduced in NAI-treated cases (odds ratio 0.36, 95% confidence interval: 0.16-0.83). Treatment within 48 hours of illness onset was significantly associated with survival (P = .04). Cases with NAI treatment initiated earlier in illness were less likely to die. Prompt treatment with NAIs may improve survival of children critically ill with influenza. Recent decreased frequency of NAI treatment of influenza may be placing untreated critically ill children at an increased risk of death.

Human Infection with Eurasian Avian-like Influenza A(H1N1) Virus, China

Human Infection with Eurasian Avian-like Influenza A(H1N1) Virus, China

Mutations at the monomer–monomer interface away from the active site of influenza B virus neuraminidase reduces susceptibility to neuraminidase inhibitor drugs

Amino acid changes in or near the active site of neuraminidase (NA) in influenza viruses reduce the susceptibility to NA inhibitor drugs. Here, we report the recovery of three influenza B viruses with reduced susceptibilities to NA inhibitors from human patients with no history of antiviral drug treatment. The three viruses were isolated by inoculating Madin-Darby canine kidney (MDCK) cells with respiratory specimens from the patients. NA inhibition assays demonstrated that two of the three isolates showed a highly reduced susceptibility to peramivir and moderately reduced susceptibility to oseltamivir, zanamivir, and laninamivir. The remaining one isolate exhibited moderately reduced sensitivity to peramivir, zanamivir, and laninamivir but was susceptible to oseltamivir. A sequence analysis of viruses propagated in MDCK cells revealed that all three isolates contained a single mutation (Q138R, P139S, or G140R) in NA not previously associated with reduced susceptibility to NA inhibitors. However, pyrosequencing analyses showed that the Q138R and G140R mutations were below a detectable level in the original clinical specimens; the P139S mutation was detected at a very low level, suggesting that the mutant viruses may be preferably selected during propagation in MDCK cells. The NA crystallographic structure showed that these mutations were located at the interface between the two monomers of the NA tetramer, away from the NA active site. In addition to amino acid substitutions around the active site of NA, these observations suggest that alterations in the monomer-monomer interface region of NA may contribute to reduced sensitivity to NA inhibitors.