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

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

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

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

Molecular distribution of amino acid substitutions on neuraminidase from the 2009 (H1N1) human influenza pandemic virus

The pandemic influenza AH1N1 (2009) caused an outbreak of human infection that spread to the world. Neuraminidase (NA) is an antigenic surface glycoprotein, which is essential to the influenza infection process, and is the target of anti-flu drugs oseltamivir and zanamivir. Currently, NA inhibitors are the pillar pharmacological strategy against seasonal and global influenza. Although mutations observed after NA-inhibitor treatment are characterized by changes in conserved amino acids of the enzyme catalytic site, it is possible that specific amino acid substitutions (AASs) distant from the active site such as H274Y, could confer oseltamivir or zanamivir resistance. To better understand the molecular distribution pattern of NA AASs, we analyzed NA AASs from all available reported pandemic AH1N1 NA sequences, including those reported from America, Africa, Asia, Europe, Oceania, and specifically from Mexico. The molecular distributions of the AASs were obtained at the secondary structure domain level for both the active and catalytic sites, and compared between geographic regions. Our results showed that NA AASs from America, Asia, Europe, Oceania and Mexico followed similar molecular distribution patterns. The compiled data of this study showed that highly conserved amino acids from the NA active site and catalytic site are indeed being affected by mutations. The reported NA AASs follow a similar molecular distribution pattern worldwide. Although most AASs are distributed distantly from the active site, this study shows the emergence of mutations affecting the previously conserved active and catalytic site. A significant number of unique AASs were reported simultaneously on different continents.     

Neuraminidase inhibitor-resistant recombinant A/Vietnam/1203/04 (H5N1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo

Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC(50)] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC(50) increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V(max) and K(m)) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.     

2010?2011年全球季节性H3N2流感病毒表面蛋白基因的分子遗传特性分析

[目的]分析2010年1月至2011年9月间全球季节性H3N2流感病毒血凝素(Hemagglutinin,HA)和神经氨酸酶(Neuraminidase,NA)基因的演变和分子特征,为流感病毒的防制提供分子信息依据.[方法]搜集期间季节性H3N2流感病毒HA和NA基因的完整核苷酸序列,分别绘制两基因编码序列的进化树;推导出相应的氨基酸序列,统计不同毒株间氨基酸位点差异并分析重要功能位点的变化.[结果]在136条完整的片段4和131条片段6中,2条HA和l条NA序列源自猪群流感病毒,剩余的序列根据进化特征可被分为两群.相比疫苗毒株,发生在HA和NA蛋白抗原位点的平均差异数分别为5.33和2.01个,3个毒株分别在HA宿主受体结合位点和二硫键及NA耐药位点出现突变,多数毒株的糖基化位点增多.江苏毒株和广东毒株分别属于群l和群2,且两省毒株间在HA蛋白抗原位点的差异数从7到13个不等.[结论]2010年1月至2011年9月间的全球季节性H3N2病毒主要呈现两种基因进化特征.因抗原性差异对疫苗开发具有指导作用,而多数毒株的抗原性检测信息仍然未知,但从抗原位点和糖基化位点的变异情况来看,多数毒株的抗原性可能已经变化,为判断是否形成新的流行株,应开展进一步的抗原性检测;并且各地区卫生行政部门应根据耐药位点的变化,制定相应的抗病毒治疗措施.     

Computational studies of H5N1 influenza virus resistance to oseltamivir

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

Compensatory Hemagglutinin Mutations Alter Antigenic Properties of Influenza Viruses

Influenza viruses routinely acquire mutations in antigenic sites on the globular head of the hemagglutinin (HA) protein. Since these antigenic sites are near the receptor binding pocket of HA, many antigenic mutations simultaneously alter the receptor binding properties of HA. We previously reported that a K165E mutation in the Sa antigenic site of A/Puerto Rico/8/34 (PR8) HA is associated with secondary neuraminidase (NA) mutations that decrease NA activity. Here, using reverse genetics, we show that the K165E HA mutation dramatically decreases HA binding to sialic acid receptors on cell surfaces. We sequentially passaged reverse-genetics-derived PR8 viruses with the K165E antigenic HA mutation in fertilized chicken eggs, and to our surprise, viruses with secondary NA mutations did not emerge. Instead, viruses with secondary HA mutations emerged in 3 independent passaging experiments, and each of these mutations increased HA binding to sialic acid receptors. Importantly, these compensatory HA mutations were located in the Ca antigenic site and prevented binding of Ca-specific monoclonal antibodies. Taken together, these data indicate that HA antigenic mutations that alter receptor binding avidity can be compensated for by secondary HA or NA mutations. Antigenic diversification of influenza viruses can therefore occur irrespective of direct antibody pressure, since compensatory HA mutations can be located in distinct antibody binding sites.     

Development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir

Determination of the sensitivity of influenza viruses to neuraminidase (NA) inhibitors is presently based on assays of NA function because, unlike available cell culture methods, the results of such assays are predictive of susceptibility in vivo. At present the most widely used substrate in assays of NA function is the fluorogenic reagent 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (MUN). A rapid assay with improved sensitivity is required because a proportion of clinical isolates has insufficient NA to be detectable in the current fluorogenic assay, and because some mutations associated with resistance to NA inhibitors reduce the activity of the enzyme. A chemiluminescence-based assay of NA activity has been developed that uses a 1,2-dioxetane derivative of sialic acid (NA-STAR) as the substrate. When compared with the fluorogenic assay, use of the NA-STAR substrate results in a 67-fold reduction in the limit of detection of the NA assay, from 200 pM (11 fmol) NA to 3 pM (0.16 fmol) NA. A panel of isolates from phase 2 clinical studies of zanamivir, which were undetectable in the fluorogenic assay, was tested for activity using the NA-STAR substrate. Of these 12 isolates with undetectable NA activity, 10 (83%) were found to have detectable NA activity using the NA-STAR substrate. A comparison of sensitivity to zanamivir of a panel of influenza A and B viruses using the two NA assay methods has been performed. IC(50) values for zanamivir using the NA-STAR were in the range 1.0-7.5 nM and those for the fluorogenic assay in the range 1. 0-5.7 nM (n = 6). The NA-STAR assay is a highly sensitive, rapid assay of influenza virus NA activity that is applicable to monitoring the susceptibility of influenza virus clinical isolates to NA inhibitors.     

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

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

Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses

If highly pathogenic H5N1 influenza viruses acquire affinity for human rather than avian respiratory epithelium, will their susceptibility to neuraminidase (NA) inhibitors (the likely first line of defense against an influenza pandemic) change as well? Adequate pandemic preparedness requires that this question be answered. We generated and tested 31 recombinants of A/Vietnam/1203/04 (H5N1) influenza virus carrying single, double, or triple mutations located within or near the receptor binding site in the hemagglutinin (HA) glycoprotein that alter H5 HA binding affinity or specificity. To gain insight into how combinations of HA and NA mutations can affect the sensitivity of H5N1 virus to NA inhibitors, we also rescued viruses carrying the HA changes together with the H274Y NA substitution, which was reported to confer resistance to the NA inhibitor oseltamivir. Twenty viruses were genetically stable. The triple N158S/Q226L/N248D HA mutation (which eliminates a glycosylation site at position 158) caused a switch from avian to human receptor specificity. In cultures of differentiated human airway epithelial (NHBE) cells, which provide an ex vivo model that recapitulates the receptors in the human respiratory tract, none of the HA-mutant recombinants showed reduced susceptibility to antiviral drugs (oseltamivir or zanamivir). This finding was consistent with the results of NA enzyme inhibition assay, which appears to predict influenza virus susceptibility in vivo. Therefore, acquisition of human-like receptor specificity does not affect susceptibility to NA inhibitors. Sequence analysis of the NA gene alone, rather than analysis of both the NA and HA genes, and phenotypic assays in NHBE cells are likely to adequately identify drug-resistant H5N1 variants isolated from humans during an outbreak.     

Analysis of oseltamivir resistance substitutions in influenza virus glycoprotein neuraminidase using a lentivirus-based surrogate assay system

Influenza A virus poses a great threat to global health, and oseltamivir (trade marked as Tamiflu), which targets influenza surface glycoprotein neuraminidase (NA), is used clinically as a major anti-influenza treatment. However, certain substitutions in NA can render an influenza virus resistant to this drug. In this study, using a lentiviral pseudotyping system, which alleviates the safety concerns of studying highly pathogenic influenza viruses such as avian influenza H5N1, that utilizes influenza surface glycoproteins (hemagglutinin or HA, and NA) and an HIV-core combined with a luciferase reporter gene as a surrogate assay, we first assessed the functionality of NA by measuring pseudovirion release in the absence or presence of oseltamivir. We demonstrated that oseltamivir displays a dose-dependent inhibition on NA activity. In contrast, a mutant NA (H274Y) is more resistant to oseltamivir treatment. In addition, the effects of several previously reported substitution NA mutants were examined as well. Our results demonstrate that this lentivirus-based pseudotyping system provides a quick, safe, and effective way to assess resistance to neuraminidase inhibitors. And we believe that as new mutations appear in influenza isolates, their impact on the effectiveness of current and future anti-NA can be quickly and reliably evaluated by this assay.     

Molecular and genetic analysis of influenza A viruses isolated in Russia, based on the neuraminidase and M2 protein gene sequence

The results of molecular analysis of 15 influenza A(H3N2) and 17-A(H1N1) epidemic strains isolated in the Russian Federation in 1995-2007 are described. The analysis on the M2 and neuraminidase influenza A virus genes was performed. The M2 sequences analysis among the remantadin resistant viruses demonstrated the S31N substitution in all strains. Besides S31N substitution, additional mutations were detected in both proteins. Mutations associated with S31N substitution were detected in each virus subtype, which may be considered as new markers for the identification of remantadin-resistant strains. The sequencing of the NA segments from all viruses showed no amino acid substitutions known to cause resistance to neuraminidase inhibitors, which indicates susceptibility to NA inhibitors among the strains.     

Pseudovirus-based neuraminidase inhibition assays reveal potential H5N1 drug-resistant mutations

The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. F or a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identifi ed to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.     

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

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

Envelope Proteins Pertain with Evolution and Adaptive Mechanism of the Novel Influenza A/H1N1 in Humans

The novel swine-origin influenza A/H1N1 virus (S-OIV) first detected in April 2009 has been identified to transmit from human to human directly and is the cause of currently emerged pandemic. In this study, nucleotide and deduced amino acid sequences of hemagglutinin (HA) and neuraminidase (NA) of the S-OIV and other influenza A viruses were analyzed through bioinformatic tools for phylogenetic analysis, genetic recombination and point mutation to investigate the emergence and adaptation of the S-OIV in human. The phylogenetic analysis showed that the HA comes from triple reassortant influenza A/H1N2 and the NA from Eurasian swine influenza A/H1N1 indicating HA and NA to descend from different lineages during the genesis of the S-OIV. Recombination analysis nullified the possibility of occurrence of recombination in HA and NA denoting the role of reassortment in the outbreak. Several conservative mutations are observed in the amino acid sequences of the HA and NA and this mutated residues are identical in the S-OIV. The results reported herein suggested the notion that the recent pandemic is the result of reassortment of different genes from different lineages of two envelope proteins, HA and NA which are responsible for antigenic activity of virus. This study further suggests that the adaptive capability of the S-OIV in human is acquired by the unique mutations generated during emergence.     

Molecular Surveillance of Antiviral Drug Resistance of Influenza A/H3N2 Virus in Singapore, 2009-2013

Adamantanes and neuraminidase inhibitors (NAIs) are two classes of antiviral drugs available for the chemoprophylaxis and treatment of influenza infections. To determine the frequency of drug resistance in influenza A/H3N2 viruses in Singapore, large-scale sequencing of neuraminidase (NA) and matrix protein (MP) genes was performed directly without initial culture amplification. 241 laboratory-confirmed influenza A/H3N2 clinical samples, collected between May 2009 and November 2013 were included. In total, 229 NA (95%) and 241 MP (100%) complete sequences were obtained. Drug resistance mutations in the NA and MP genes were interpreted according to published studies. For the NAIs, a visual inspection of the aligned NA sequences revealed no known drug resistant genotypes (DRGs). For the adamantanes, the well-recognised S31N DRG was identified in all 241 MP genes. In addition, there was an increasing number of viruses carrying the combination of D93G+Y155F+D251V (since May 2013) or D93G (since March 2011) mutations in the NA gene. However, in-vitro NAI testing indicated that neither D93G+Y155F+D251V nor D93G alone conferred any changes in NAI susceptibility. Lastly, an I222T mutation in the NA gene that has previously been reported to cause oseltamivir-resistance in influenza A/H1N1/2009, B, and A/H5N1, was detected from a treatment-naïve patient. Further in-vitro NAI testing is required to confirm the effect of this mutation in A/H3N2 virus.     

In silico prediction of drug resistance due to S247R mutation of Influenza H1N1 neuraminidase protein

We present here in silico studies on antiviral drug resistance due to a novel mutation of influenza A/H1N1 neuraminidase (NA) protein. Influenza A/H1N1 virus was responsible for a recent pandemic and is currently circulating among the seasonal influenza strains. M2 and NA are the two major viral proteins related to pathogenesis in humans and have been targeted for drug designing. Among them, NA is preferred because the ligand-binding site of NA is highly conserved between different strains of influenza virus. Different mutations of the NA active site residues leading to drug resistance or susceptibility of the virus were studied earlier. We report here a novel mutation (S247R) in the NA protein that was sequenced earlier from the nasopharyngeal swab from Sri Lanka and Thailand in the year 2009 and 2011, respectively. Another mutation (S247N) was already known to confer resistance to oseltamivir. We did a comparative study of these two mutations vis-a-vis the drug-sensitive wild type NA to understand the mechanism of drug resistance of S247N and to predict the probability of the novel S247R mutation to become resistant to the currently available drugs, oseltamivir and zanamivir. We performed molecular docking- and molecular dynamics-based analysis of both the mutant proteins and showed that mutation of S247R affects drug binding to the protein by positional displacement due to altered active site cavity architecture, which in turn reduces the affinity of the drug molecules to the NA active site. Our analysis shows that S247R may have high probability of being resistant.     

Distinct glycoprotein inhibitors of influenza A virus in different animal sera.

Normal horse and guinea pig sera contain the glycoprotein inhibitor alpha 2-macroglobulin, which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. In the current study, the presence of inhibitors of influenza A virus in pig and rabbit sera was investigated. Variants of influenza virus type A/Los Angeles/2/87(H3N2) that were resistant to horse, pig, or rabbit serum were isolated. Analysis of the variant viruses with anti-hemagglutinin (HA) monoclonal antibodies revealed that antigenic changes occurred with the development of serum inhibitor resistance. Characterization of the inhibitors in pig and rabbit sera by using periodate and receptor-destroying enzyme demonstrated that carbohydrate is an important constituent of the active portion of both inhibitor molecules and that sialic acid is involved in the interaction of the inhibitors with influenza virus HA. Nucleotide sequence analysis of the HA molecule revealed that the serum-resistant variants each acquired a different set of amino acid alterations. The multiply resistant variants maintained the original amino acid changes and acquired additional changes. Sequence modifications in the HA involved the conserved amino acids within the receptor binding site (RBS) at position 137 and the second-shell RBS residues at positions 155 and 186. Amino acid changes also occurred within antigenic site A (position 145) and directly behind the receptor binding pocket (position 220). Amino acid alterations resulted in the acquisition of a potential glycosylation site at position 128 and the loss of potential glycosylation sites at positions 246 and 248. The localization of the amino acid changes in HA1 to the region of the RBS supports the concept of serum inhibitors as receptor analogs. The unique set of mutations acquired by the serum inhibitor-resistant variants strongly suggests that horse, pig, and rabbit sera each contain distinct glycoprotein inhibitors of influenza A virus.     

Systematic Identification of H274Y Compensatory Mutations in Influenza A Virus Neuraminidase by High-Throughput Screening

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.     

A novel pathogenic mechanism of highly pathogenic avian influenza H5N1 viruses involves hemagglutinin mediated resistance to serum innate inhibitors

In this study, the effect of innate serum inhibitors on influenza virus infection was addressed. Seasonal influenza A(H1N1) and A(H3N2), 2009 pandemic A(H1N1) (H1N1pdm) and highly pathogenic avian influenza (HPAI) A(H5N1) viruses were tested with guinea pig sera negative for antibodies against all of these viruses as evaluated by hemagglutination-inhibition and microneutralization assays. In the presence of serum inhibitors, the infection by each virus was inhibited differently as measured by the amount of viral nucleoprotein produced in Madin-Darby canine kidney cells. The serum inhibitors inhibited seasonal influenza A(H3N2) virus the most, while the effect was less in seasonal influenza A(H1N1) and H1N1pdm viruses. The suppression by serum inhibitors could be reduced by heat inactivation or treatment with receptor destroying enzyme. In contrast, all H5N1 strains tested were resistant to serum inhibitors. To determine which structure (hemagglutinin (HA) and/or neuraminidase (NA)) on the virus particles that provided the resistance, reverse genetics (rg) was applied to construct chimeric recombinant viruses from A/Puerto Rico/8/1934(H1N1) (PR8) plasmid vectors. rgPR8-H5 HA and rgPR8-H5 HANA were resistant to serum inhibitors while rgPR8-H5 NA and PR8 A(H1N1) parental viruses were sensitive, suggesting that HA of HPAI H5N1 viruses bestowed viral resistance to serum inhibition. These results suggested that the ability to resist serum inhibition might enable the viremic H5N1 viruses to disseminate to distal end organs. The present study also analyzed for correlation between susceptibility to serum inhibitors and number of glycosylation sites present on the globular heads of HA and NA. H3N2 viruses, the subtype with highest susceptibility to serum inhibitors, harbored the highest number of glycosylation sites on the HA globular head. However, this positive correlation cannot be drawn for the other influenza subtypes.