Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.     

Birds and Viruses at a Crossroad - Surveillance of Influenza A Virus in Portuguese Waterfowl

During recent years, extensive amounts of data have become available regarding influenza A virus (IAV) in wild birds in northern Europe, while information from southern Europe is more limited. Here, we present an IAV surveillance study conducted in western Portugal 2008–2009, analyzing 1653 samples from six different species of waterfowl, with the majority of samples taken from Mallards (Anas platyrhynchos). Overall 4.4% of sampled birds were infected. The sampling results revealed a significant temporal variation in the IAV prevalence, including a pronounced peak among predominantly young birds in June, indicating that IAV circulate within breeding populations in the wetlands of western Portugal. The H10N7 and H9N2 subtypes were predominant among isolated viruses. Phylogenetic analyses of the hemagglutinin and neuraminidase sequences of H10N7, H9N2 and H11N3 virus showed that sequences from Portugal were closely related to viral sequences from Central Europe as well as to IAVs isolated in the southern parts of Africa, reflecting Portugal’s position on the European-African bird migratory flyway. This study highlights the importance of Portugal as a migratory crossroad for IAV, connecting breeding stationary waterfowl with birds migrating between continents which enable transmission and spread of IAV.     

Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.     

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.     

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.     

Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam

Background

Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses.

Methods and Findings

Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found.

Conclusion

In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.     

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.     

Genomic Characterization of H14 Subtype Influenza A Viruses in New World Waterfowl and Experimental Infectivity in Mallards (Anas platyrhynchos)

Recent repeated isolation of H14 hemagglutinin subtype influenza A viruses (IAVs) in the New World waterfowl provides evidence to suggest that host and/or geographic ranges for viruses of this subtype may be expanding. In this study, we used genomic analyses to gain inference on the origin and evolution of H14 viruses in New World waterfowl and conducted an experimental challenge study in mallards (Anas platyrhynchos) to evaluate pathogenicity, viral replication, and transmissibility of a representative viral strain in a natural host species. Genomic characterization of H14 subtype IAVs isolated from New World waterfowl, including three isolates sequenced specifically for this study, revealed high nucleotide identity among individual gene segments (e.g. ≥95% shared identity among H14 HA gene segments). In contrast, lower shared identity was observed among internal gene segments. Furthermore, multiple neuraminidase subtypes were observed for H14 IAVs isolated in the New World. Gene segments of H14 viruses isolated after 2010 shared ancestral genetic lineages with IAVs isolated from wild birds throughout North America. Thus, genomic characterization provided evidence for viral evolution in New World waterfowl through genetic drift and genetic shift since purported introduction from Eurasia. In the challenge study, no clinical disease or lesions were observed among mallards experimentally inoculated with A/blue-winged teal/Texas/AI13-1028/2013(H14N5) or exposed via contact with infected birds. Titers of viral shedding for mallards challenged with the H14N5 IAV were highest at two days post-inoculation (DPI); however shedding was detected up to nine DPI using cloacal swabs. The distribution of viral antigen among mallards infected with H14N5 IAV was largely restricted to enterocytes lining the villi in the lower intestinal tract and in the epithelium of the bursa of Fabricius. Characterization of the infectivity of A/blue-winged teal/Texas/AI13-1028/2013(H14N5) in mallards provides support for similarities in viral replication and shedding as compared to previously described waterfowl-adapted, low pathogenic IAV strains in ducks.     

Triple Combination of Amantadine,Ribavirin, and Oseltamivir Is Highly Active and Synergistic against Drug Resistant Influenza Virus Strains In Vitro

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21^st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza.     

Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses

An epidemic of an avian-origin H7N9 influenza virus has recently emerged in China, infecting 134 patients of which 45 have died. This is the first time that an influenza virus harboring an N9 serotype neuraminidase (NA) has been known to infect humans. H7N9 viruses are divergent and at least two distinct NAs and hemagglutinins (HAs) have been found, respectively, from clinical isolates. The prototypes of these viruses are A/Anhui/1/2013 and A/Shanghai/1/2013. NAs from these two viruses are distinct as the A/Shanghai/1/2013 NA has an R294K substitution that can confer NA inhibitor oseltamivir resistance. Oseltamivir is by far the most commonly used anti-influenza drug due to its potency and high bioavailability. In this study, we show that an R294K substitution results in multidrug resistance with extreme oseltamivir resistance (over 100 000-fold) using protein- and virus-based assays. To determine the molecular basis for the inhibitor resistance, we solved high-resolution crystal structures of NAs from A/Anhui/1/2013 N9 (R294-containing) and A/Shanghai/1/2013 N9 (K294-containing). R294K substitution results in an unfavorable E276 conformation for oseltamivir binding, and consequently loss of inhibitor carboxylate interactions, which compromises the binding of all classical NA ligands/inhibitors. Moreover, we found that R294K substitution results in reduced NA catalytic efficiency along with lower viral fitness. This helps to explain why K294 has predominantly been found in clinical cases of H7N9 infection under the selective pressure of oseltamivir treatment and not in the dominant human-infecting viruses. This implies that oseltamivir can still be efficiently used in the treatment of H7N9 infections.     

Are ducks contributing to the endemicity of highly pathogenic H5N1 influenza virus in Asia?

Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to na?ve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.     

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.     

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).     

The fight against new types of influenza virus

In 1997, during an outbreak in chickens in Hong Kong the avian H5N1 influenza virus crossed the species barrier and infected 18 people, of which 6 cases were fatal. The virus also infected wild birds and continued to circulate and mutate in geese and ducks in southeastern China. Since this occurrence, new antigenic variants that are highly pathogenic for humans as well as wild, domestic, and exotic waterfowl continue to appear in Hong Kong. This virus is spreading across Asia, and is encroaching upon Europe and other continents. Wild birds are now considered as the main reservoir of H5N1 virus. Humans become infected with this H5N1 virus usually via close contact with infected birds or a highly contaminated environment. The very low transmissibility of this virus prevented further person-to-person dissemination in spite of the complete absence of immunity in the human population to H5N1 viruses. Viruses of the H5N1 subtype are characterized by an exceptionally high pathogenicity for humans. The cause of the viral virulence is not known so far; however, several virulence factors are considered. The unprecedented capability of H5N1 viruses to kill humans intensifies the concern about its pandemic potential with catastrophic consequences. The effectiveness of existing antivirals as well as vaccines for humans and birds are reviewed.     

Influence of body condition on influenza A virus infection in mallard ducks: experimental infection data

Migrating waterfowl are implicated in the global spread of influenza A viruses (IAVs), and mallards (Anas platyrhynchos) are considered a particularly important IAV reservoir. Prevalence of IAV infection in waterfowl peaks during autumn pre-migration staging and then declines as birds reach wintering areas. Migration is energetically costly and birds often experience declines in body condition that may suppress immune function. We assessed how body condition affects susceptibility to infection, viral shedding and antibody production in wild-caught and captive-bred juvenile mallards challenged with low pathogenic avian influenza virus (LPAIV) H5N9. Wild mallards (n = 30) were separated into three experimental groups; each manipulated through food availability to a different condition level (−20%, −10%, and normal ±5% original body condition), and captive-bred mallards (n = 10) were maintained at normal condition. We found that wild mallards in normal condition were more susceptible to LPAIV infection, shed higher peak viral loads and shed viral RNA more frequently compared to birds in poor condition. Antibody production did not differ according to condition. We found that wild mallards did not differ from captive-bred mallards in viral intensity and duration of infection, but they did exhibit lower antibody titers and greater variation in viral load. Our findings suggest that reduced body condition negatively influences waterfowl host competence to LPAIV infection. This observation is contradictory to the recently proposed condition-dependent hypothesis, according to which birds in reduced condition would be more susceptible to IAV infection. The mechanisms responsible for reducing host competency among birds in poor condition remain unknown. Our research indicates body condition may influence the maintenance and spread of LPAIV by migrating waterfowl.     

Environmental levels of the antiviral oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC₅₀ for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.     

Evidence of infection by H5N2 highly pathogenic avian influenza viruses in healthy wild waterfowl

The potential existence of a wild bird reservoir for highly pathogenic avian influenza (HPAI) has been recently questioned by the spread and the persisting circulation of H5N1 HPAI viruses, responsible for concurrent outbreaks in migratory and domestic birds over Asia, Europe, and Africa. During a large-scale surveillance programme over Eastern Europe, the Middle East, and Africa, we detected avian influenza viruses of H5N2 subtype with a highly pathogenic (HP) viral genotype in healthy birds of two wild waterfowl species sampled in Nigeria. We monitored the survival and regional movements of one of the infected birds through satellite telemetry, providing a rare evidence of a non-lethal natural infection by an HP viral genotype in wild birds. Phylogenetic analysis of the H5N2 viruses revealed close genetic relationships with H5 viruses of low pathogenicity circulating in Eurasian wild and domestic ducks. In addition, genetic analysis did not reveal known gallinaceous poultry adaptive mutations, suggesting that the emergence of HP strains could have taken place in either wild or domestic ducks or in non-gallinaceous species. The presence of coexisting but genetically distinguishable avian influenza viruses with an HP viral genotype in two cohabiting species of wild waterfowl, with evidence of non-lethal infection at least in one species and without evidence of prior extensive circulation of the virus in domestic poultry, suggest that some strains with a potential high pathogenicity for poultry could be maintained in a community of wild waterfowl.     

Outside‐binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1

The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA‐oseltamivir complex (PDB ID: 3NSS) was used as a wild‐type structure. After selecting the target NA sequences, their three‐dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free‐energy analysis using the MM‐PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM‐PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature. © 2012 Wiley Periodicals, Inc.     

Oseltamivir-Resistant Variants of the 2009 Pandemic H1N1 Influenza A Virus Are Not Attenuated in the Guinea Pig and Ferret Transmission Models

Oseltamivir is routinely used worldwide for the treatment of severe influenza A virus infection, and should drug-resistant pandemic 2009 H1N1 viruses become widespread, this potent defense strategy might fail. Oseltamivir-resistant variants of the pandemic 2009 H1N1 influenza A virus have been detected in a substantial number of patients, but to date, the mutant viruses have not moved into circulation in the general population. It is not known whether the resistance mutations in viral neuraminidase (NA) reduce viral fitness. We addressed this question by studying transmission of oseltamivir-resistant mutants derived from two different isolates of the pandemic H1N1 virus in both the guinea pig and ferret transmission models. In vitro, the virus readily acquired a single histidine-to-tyrosine mutation at position 275 (H275Y) in viral neuraminidase when serially passaged in cell culture with increasing concentrations of oseltamivir. This mutation conferred a high degree of resistance to oseltamivir but not zanamivir. Unexpectedly, in guinea pigs and ferrets, the fitness of viruses with the H275Y point mutation was not detectably impaired, and both wild-type and mutant viruses were transmitted equally well from animals that were initially inoculated with 1:1 virus mixtures to naïve contacts. In contrast, a reassortant virus containing an oseltamivir-resistant seasonal NA in the pandemic H1N1 background showed decreased transmission efficiency and fitness in the guinea pig model. Our data suggest that the currently circulating pandemic 2009 H1N1 virus has a high potential to acquire drug resistance without losing fitness.Oseltamivir resistance was rare until 2008, when resistant seasonal H1N1 viruses were found circulating in the general Scandinavian population (15). Soon after, studies from other countries in Europe also reported the isolation of oseltamivir-resistant viruses, and eventually, oseltamivir resistance was recognized as a global phenomenon (9, 27). Prior to 2008, resistant viruses were primarily isolated from patients with nonresponsive influenza virus infections or from infected patients who received a low-dose prophylaxis regiment prior to symptom onset. At the time, these resistant isolates accounted for 1% of the circulating H1N1 virus. Drug resistance mutations were identified during oseltamivir development, including a histidine-to-tyrosine mutation at position 275 (H275Y) in N1 neuraminidase (NA). This mutation in particular was shown to attenuate virus growth and pathology in ferrets (17). Additionally, oseltamivir-resistant viruses with a nearby mutation in N2 neuraminidase transmitted less efficiently than oseltamivir-sensitive viruses in the guinea pig transmission model (4). Surprisingly, the seasonal 2008 H1N1 viral isolates that spread around the world had the same tyrosine mutation, which was previously associated with iatrogenic infections and attenuation. Furthermore, epidemiological studies concluded that this resistant virus developed independently of drug selection, suggesting that compensatory adaptations allowed an attenuating mutation to become permissible (3, 18). The ability of resistant 2008 isolates to perform on par with nonresistant 2008 isolates in growth curves, in mean plaque size, and in a transmission model was recently confirmed (2). Currently, 99% of seasonal H1N1 viruses are oseltamivir resistant; however, the prevalence of these viruses is very low due to replacement by a novel reassortant H1N1 virus (6, 8). This novel reassortant was originally identified in Mexico by doctors concerned about a jump in the number of influenza cases during the month of March in 2009 (7). Later referred to as swine-origin influenza virus, novel H1N1 virus, or 2009 pandemic H1N1 virus, this virus would continue to efficiently transmit around the world, even during the summer months of the northern hemisphere. Its robust transmission was later confirmed in aerosol transmission models, in which 86% of ferrets and 100% of guinea pigs exposed to infected animals contracted pandemic influenza (22, 28, 31). Oseltamivir was used broadly during the outbreak, treating those with complications and prophylactically treating close contacts of confirmed cases. The use of oseltamivir in this manner provided ample opportunity for oseltamivir-resistant viruses to develop. More than 225 cases of oseltamivir-resistant infections have been confirmed from the beginning of the pandemic, including four incidents of suspected aerosol transmission (21, 32, 33). Fortunately, these clinical isolates never progressed into stable transmission in the general public. This study seeks to evaluate if introducing the H275Y mutation into the pandemic 2009 H1N1 virus attenuates virus replication in vitro or in vivo using the guinea pig model and the ferret model to test aerosol transmission efficiency. Furthermore, this study evaluates if a reassortant between the circulating novel H1N1 virus and seasonal neuraminidase (NA) forms a well-adapted, resistant virus capable of efficient transmission.Currently, oseltamivir is the drug of choice for treating novel H1N1 complications and outpatient prophylaxis. Therefore, it is of great importance to study the in vitro replication and transmission phenotypes of oseltamivir-resistant novel H1N1 viruses to understand why broad oseltamivir resistance has not occurred or whether we should expect it to occur in the future.