Effect of an asparagine-to-serine mutation at position 294 in neuraminidase on the pathogenicity of highly pathogenic H5N1 influenza A virus

Like the histidine-to-tyrosine substitution at position 274 in neuraminidase (NA H274Y), an asparagine-to-serine mutation at position 294 in this protein (NA N294S) confers oseltamivir resistance to highly pathogenic H5N1 influenza A viruses. However, unlike viruses with the NA H274Y mutation, the properties of viruses possessing NA N294S are not well understood. Here, we assessed the effect of the NA N294S substitution on the replication and pathogenicity of human H5N1 viruses and on the efficacy of the NA inhibitors oseltamivir and zanamivir in mouse and ferret models. Although NA N294S-possessing H5N1 viruses were attenuated in mice and ferrets compared to their oseltamivir-sensitive counterparts, one of the infected ferrets died from systemic infection, demonstrating the potential lethality in ferrets of oseltamivir-resistant H5N1 viruses with the NA N294S substitution. The efficacy of oseltamivir, but not that of zanamivir, against an NA N294S-possessing virus was substantially impaired both in ferrets and in vitro. These results demonstrate the considerable pathogenicity of NA N294S substitution-possessing H5N1 viruses and underscore the importance of monitoring the emergence of the NA N294S mutation in circulating H5N1 viruses.     

Phylogenetic and genetic characterization of a 2017 clinical isolate of H7N9 virus in Guangzhou,China during the fifth epidemic wave

Pathogenic H7N9 influenza viruses continue to pose a public health concern. The H7N9 virus has caused five outbreak waves of human infections in China since 2013. In the present study, a novel H7N9 strain (A/Guangdong/8H324/2017) was isolated from a female patient with severe respiratory illness during the fifth wave of the 2017 H7N9 epidemic. Phylogenetic analysis showed that the H7N9 viruses collected during the fifth wave belong to two different lineages: the Pearl River Delta lineage and the Yangtze River Delta lineage. The novel isolate is closely related to the Pearl River Delta H7N9 viruses, which were isolated from patients in Guangdong Province. The novel H7N9 isolate has an insertion of three basic amino acids in the cleavage site of hemagglutinin (HA), which may enhance virulence in poultry. The 2017 isolate also possesses an R292K substitution in the neuraminidase (NA) protein, which confers oseltamivir resistance. This study highlights the pandemic potential of the novel H7N9 virus in mammals; thus, future characterization and surveillance is warranted.     

Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure

Background

Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied.

Aim and Methods

In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naïve Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype.

Results and Conclusion

Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.     

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).     

Characterization of Oseltamivir-Resistant 2009 H1N1 Pandemic Influenza A Viruses

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.     

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.     

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.     

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

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

Functional and Structural Analysis of Influenza Virus Neuraminidase N3 Offers Further Insight into the Mechanisms of Oseltamivir Resistance

The influenza virus neuraminidase H274Y substitution is a highly prevalent amino acid substitution associated with resistance to the most heavily used influenza drug, oseltamivir. Previous structural studies suggest that the group specific 252 residue (Y252 in group 1 and T252 in group 2) might be a key factor underlying H274Y resistance. However, H274Y has only been reported in N1 subtypes, which indicates that there must be additional key residues that determine H274Y resistance. Furthermore, we found that members of NA serotype N3 also possess Y252, raising the key question as to whether or not H274Y resistance may also be possible for some group 2 NAs. Here, we demonstrate that the H274Y substitution results in mild oseltamivir resistance for N3. Comparative structural analysis of N3, N1, and their 274Y variants indicates that the interaction of residue 296 (H in N1 and nonaromatic for other serotypes) with conserved W295 is another important determinant of oseltamivir resistance.     

Competitive Fitness of Oseltamivir-Sensitive and -Resistant Highly Pathogenic H5N1 Influenza Viruses in a Ferret Model

The fitness of oseltamivir-resistant highly pathogenic H5N1 influenza viruses has important clinical implications. We generated recombinant human A/Vietnam/1203/04 (VN; clade 1) and A/Turkey/15/06 (TK; clade 2.2) influenza viruses containing the H274Y neuraminidase (NA) mutation, which confers resistance to NA inhibitors, and compared the fitness levels of the wild-type (WT) and resistant virus pairs in ferrets. The VN-H274Y and VN-WT viruses replicated to similar titers in the upper respiratory tract (URT) and caused comparable disease signs, and none of the animals survived. On days 1 to 3 postinoculation, disease signs caused by oseltamivir-resistant TK-H274Y virus were milder than those caused by TK-WT virus, and all animals survived. We then studied fitness by using a novel approach. We coinoculated ferrets with different ratios of oseltamivir-resistant and -sensitive H5N1 viruses and measured the proportion of clones in day-6 nasal washes that contained the H274Y NA mutation. Although the proportion of VN-H274Y clones increased consistently, that of TK-H274Y virus decreased. Mutations within NA catalytic (R292K) and framework (E119A/K, I222L, H274L, and N294S) sites or near the NA enzyme active site (V116I, I117T/V, Q136H, K150N, and A250T) emerged spontaneously (without drug pressure) in both pairs of viruses. The NA substitutions I254V and E276A could exert a compensatory effect on the fitness of VN-H274Y and TK-H274Y viruses. NA enzymatic function was reduced in both drug-resistant H5N1 viruses. These results show that the H274Y NA mutation affects the fitness of two H5N1 influenza viruses differently. Our novel method of assessing viral fitness accounts for both virus-host interactions and virus-virus interactions within the host.The neuraminidase (NA) inhibitors (orally administered oseltamivir and inhaled zanamivir) are currently an important class of antiviral drugs available for the treatment of seasonal and pandemic influenza. Although administration of NA inhibitors may significantly reduce influenza virus transmission, it risks the emergence of drug-resistant variants (16, 32). The impact of drug resistance would depend on the fitness (i.e., infectivity in vitro and virulence and transmissibility in vivo) of the resistant virus. If the resistance mutation only modestly reduces the virus'' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. The unexpected natural emergence and spread of oseltamivir-resistant variants (carrying the H274Y NA amino acid substitution) among seasonal H1N1 influenza viruses of the A/Brisbane/59/07 lineage demonstrated that drug-resistant viruses can be highly fit and transmissible in humans (11, 22, 29), although the fitness of these variants is not completely understood. They are hypothesized to have lower NA receptor affinity and more-optimal NA and hemagglutinin (HA) functional balance than do wild-type (WT) viruses (38). Fortunately, oseltamivir-resistant variants have rarely been reported to occur among the novel pandemic H1N1 influenza viruses that emerged in April 2009; therefore, initial data suggest that currently circulating wild-type viruses possibly possess greater fitness than drug-resistant viruses (45), although only retrospective epidemiological data can provide a conclusive answer. The key questions are whether the risk posed by NA inhibitor-resistant viruses can be assessed experimentally and what the most reliable approach may be.All NA inhibitor-resistant influenza viruses characterized to date have contained specific mutations in the NA molecule. Clinically derived drug-resistant viruses have carried mutations that are NA subtype specific and differ in accordance with the NA inhibitor used (12, 35). The most commonly observed mutations are H274Y and N294S in the influenza A N1 NA subtype, E119A/G/D/V and R292K in the N2 NA subtype, and R152K and D198N in influenza B viruses (35, 36). The fitness of NA inhibitor-resistant viruses has been studied in vitro and in vivo. Many groups have assessed their replicative capacity in MDCK cells, but this assay system can yield anomalous results (49), particularly in the case of low-passage clinical isolates. The mismatch between virus specificity and cellular receptors can be overcome by using cell lines engineered to express human-like α-2,6-linked sialyl cell surface receptors (MDCK-SIAT1) (15, 34) or a novel cell culture-based system that morphologically and functionally recapitulates differentiated normal human bronchial epithelial (NHBE) cells (24). Investigations in vivo typically compare replication efficiencies, clinical signs, and transmissibility levels between oseltamivir-resistant viruses and the corresponding wild-type virus. Initial studies found that NA inhibitor-resistant influenza viruses were severely compromised in vitro and in animal models (6, 17, 26) and thus led to the idea that resistant viruses will unlikely have an impact on epidemic and pandemic influenza. However, clinically derived H1N1 virus with the H274Y NA mutation (18) and reverse genetics-derived H3N2 virus with the E119V NA mutation (46) were subsequently found to possess biological fitness and transmissibility similar to those of drug-sensitive virus in direct-contact ferrets. Recent studies in a guinea pig model showed that recombinant human H3N2 influenza viruses carrying either a single E119V NA mutation or the double NA mutation E119V-I222V were transmitted efficiently by direct contact but not by aerosol (5).There is limited information about the fitness of NA inhibitor-resistant H5N1 influenza viruses. Although they are not efficiently transmitted from human to human, their pandemic potential remains a serious public health concern because of their virulence in humans (1, 4, 7). H5N1 viruses isolated from untreated patients are susceptible to the NA inhibitors oseltamivir and zanamivir (21), although oseltamivir-resistant variants with the H274Y NA mutation have been reported to occur in five patients after (9, 30) or before (41) treatment with oseltamivir. The World Health Organization reported the isolation of two oseltamivir-resistant H5N1 viruses from an Egyptian girl and her uncle (44) after oseltamivir treatment. The virus was moderately resistant and possessed an N294S NA mutation. Preliminary evidence suggests that the resistance mutation existed before transmission of the virus from birds to the patients and thus before initiation of treatment (41). We previously showed that wild-type A/Vietnam/1203/04 (H5N1) influenza virus and recombinants carrying either the H274Y or the N294S NA mutation reached comparable titers in MDCK and MDCK-SIAT1 cells and caused comparable mortality rates among BALB/c mice (48). In contrast, clinically derived A/Hanoi/30408/05 (H5N1) influenza virus with the H274Y NA mutation reproduced to lower titers than the oseltamivir-sensitive virus in the lungs of inoculated ferrets (30).In a ferret model, we compared the fitness levels of two pairs of H5N1 viruses in the absence of selective drug pressure. One virus of each pair was the wild type, while the other carried the H274Y NA mutation conferring oseltamivir resistance. The two viruses used, A/Vietnam/1203/04 (HA clade 1) and A/Turkey/15/06 (HA clade 2.2), differ in their pathogenicity to ferrets. Virus fitness was evaluated by two approaches. Using the traditional approach, we compared clinical disease signs, relative inactivity indexes, weight and temperature changes, and virus replication levels in the upper respiratory tract (URT). We then used a novel competitive fitness approach in which we genetically analyzed individual virus clones after coinfection of ferrets with mixtures of oseltamivir-sensitive and -resistant H5N1 viruses; thus, we determined virus-virus interactions within the host. We observed no difference between the resistant and sensitive virus of each pair in clinical signs or virus replication in the URT; however, analysis of virus-virus interactions within the host showed that the H274Y NA mutation affected the fitness of the two viruses differently. The oseltamivir-resistant A/Vietnam/1203/04-like virus outgrew its wild-type counterpart, while the oseltamivir-resistant A/Turkey/15/06-like virus showed less fitness than its wild-type counterpart.     

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.     

Efficacy of the New Neuraminidase Inhibitor CS-8958 against H5N1 Influenza Viruses

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 µg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants.     

Importance of neuraminidase active-site residues to the neuraminidase inhibitor resistance of influenza viruses

Neuraminidase inhibitors (NAIs) are antivirals designed to target conserved residues at the neuraminidase (NA) enzyme active site in influenza A and B viruses. The conserved residues that interact with NAIs are under selective pressure, but only a few have been linked to resistance. In the A/Wuhan/359/95 (H3N2) recombinant virus background, we characterized seven charged, conserved NA residues (R118, R371, E227, R152, R224, E276, and D151) that directly interact with the NAIs but have not been reported to confer resistance to NAIs. These NA residues were replaced with amino acids that possess side chains having similar properties to maintain their original charge. The NA mutations we introduced significantly decreased NA activity compared to that of the A/Wuhan/359/95 recombinant wild-type and R292K (an NA mutation frequently reported to confer resistance) viruses, which were analyzed for comparison. However, the recombinant viruses differed in replication efficiency when we serially passaged them in vitro; the growth of the R118K and E227D viruses was most impaired. The R224K, E276D, and R371K mutations conferred resistance to both zanamivir and oseltamivir, while the D151E mutation reduced susceptibility to oseltamivir only (approximately 10-fold) and the R152K mutation did not alter susceptibility to either drug. Because the R224K mutation was genetically unstable and the emergence of the R371K mutation in the N2 subtype is statistically unlikely, our results suggest that only the E276D mutation is likely to emerge under selective pressure. The results of our study may help to optimize the design of NAIs.     

Estimating the Fitness Advantage Conferred by Permissive Neuraminidase Mutations in Recent Oseltamivir-Resistant A(H1N1)pdm09 Influenza Viruses

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.     

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.     

Human H7N9 Influenza A Viruses Replicate in Swine Respiratory Tissue Explants

Recently, novel H7N9 influenza viruses have caused an unprecedented outbreak in humans. Pigs are an important intermediate host for influenza; thus, we assessed the replication ability of three human H7N9 viruses (A/Anhui/1/2013, A/Shanghai/1/2013, A/Shanghai/2/2013) in swine tissue explants. All viruses tested replicated efficiently in explants from tracheas and bronchi, with limited replication in alveolar cells. Swine respiratory tissue explants can serve as an efficient model for screening replication potential of newly emerging H7N9 viruses.     

Inhibition of viral group-1 and group-2 neuraminidases by oseltamivir: A comparative structural analysis by the ScrewFit algorithm

The viral surface glycoprotein neuraminidase (NA) allows the influenza virus penetration and the egress of virions. NAs are classified as A, B, and C. Type-A NAs from influenza virus are subdivided into two phylogenetically distinct families, group-1 and group-2. NA inhibition by oseltamivir represents a therapeutic approach against the avian influenza virus H5N1. Here, structural bases for oseltamivir recognition by group-1 NA1, NA8 and group-2 NA9 are highlighted by the ScrewFit algorithm for quantitative structure comparison. Oseltamivir binding to NA1 and NA8 affects the geometry of Glu119 and of regions Arg130-Ser160, Val240-Gly260, and Asp330-Glu382, leading to multiple NA conformations. Additionally, although NA1 and NA9 share almost the same oseltamivir-bound final conformation, they show some relevant differences as suggested by the ScrewFit algorithm. These results indicate that the design of new NA inhibitors should take into account these family-specific effects induced on the whole structure of NAs.     

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

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

Enhanced mammalian transmissibility of seasonal influenza A/H1N1 viruses encoding an oseltamivir-resistant neuraminidase

Between 2007 and 2009, oseltamivir resistance developed among seasonal influenza A/H1N1 (sH1N1) virus isolates at an exponential rate, without a corresponding increase in oseltamivir usage. We hypothesized that the oseltamivir-resistant neuraminidase (NA), in addition to being relatively insusceptible to the antiviral effect of oseltamivir, might confer an additional fitness advantage on these viruses by enhancing their transmission efficiency among humans. Here we demonstrate that an oseltamivir-resistant clinical isolate, an A/Brisbane/59/2007(H1N1)-like virus isolated in New York State in 2008, transmits more efficiently among guinea pigs than does a highly similar, contemporaneous oseltamivir-sensitive isolate. With reverse genetics reassortants and point mutants of the two clinical isolates, we further show that expression of the oseltamivir-resistant NA in the context of viral proteins from the oseltamivir-sensitive virus (a 7:1 reassortant) is sufficient to enhance transmissibility. In the guinea pig model, the NA is the critical determinant of transmission efficiency between oseltamivir-sensitive and -resistant Brisbane/59-like sH1N1 viruses, independent of concurrent drift mutations that occurred in other gene products. Our data suggest that the oseltamivir-resistant NA (specifically, one or both of the companion mutations, H275Y and D354G) may have allowed resistant Brisbane/59-like viruses to outtransmit sensitive isolates. These data provide in vivo evidence of an evolutionary mechanism that would explain the rapidity with which oseltamivir resistance achieved fixation among sH1N1 isolates in the human reservoir.     

I223R Mutation in Influenza A(H1N1)pdm09 Neuraminidase Confers Reduced Susceptibility to Oseltamivir and Zanamivir and Enhanced Resistance with H275Y

Background

Resistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility.

Methods

The NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0–15) and zanamivir (days 15–25 and 70–80).

Results

Compared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6–69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin.

Conclusions

Reduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines the importance of close monitoring of treated patients especially those immunocompromised.