The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo

Oseltamivir carboxylate is a potent and specific inhibitor of influenza A and B neuraminidase (NA). Oseltamivir phosphate, the ethyl ester prodrug of oseltamivir carboxylate, is the first orally active NA inhibitor available for the prophylaxis and treatment of influenza A and B. It offers an improvement over amantadine and rimantadine which are active only against influenza A and rapidly generate resistant virus. The emergence of virus resistant to oseltamivir carboxylate in the treatment of naturally acquired influenza infection is low (about 1%). The types of NA mutation to arise are sub-type specific and largely predicted from in vitro drug selection studies. A substitution of the conserved histidine at position 274 for tyrosine in the NA active site has been selected via site directed mutagenesis, serial passage in culture under drug pressure in H1N1 and during the treatment of experimental H1N1 infection in man. Virus carrying H274Y NA enzyme selected in vivo has reduced sensitivity to oseltamivir carboxylate. The replicative ability in cell culture was reduced up to 3 logs, as was infectivity in animal models of influenza virus infection. Additionally, pathogenicity of the mutant virus is significantly compromised in ferret, compared to the corresponding wild type virus. Virus carrying a H274Y mutation is unlikely to be of clinical consequence in man.     

Oseltamivir: a clinical and pharmacological perspective

Oseltamivir is the ethyl ester prodrug of the antiviral molecule, oseltamivir carboxylate, a potent and selective inhibitor of influenza A and B neuraminidase (NA) (sialidase). It is highly bioavailable in capsule and suspension formulations and, after conversion to the active metabolite in the liver, distributes throughout the body, including the upper and lower respiratory tract. Oseltamivir carboxylate is 3% bound to human plasma proteins and eliminated through the kidneys by a first-order process as unchanged drug by glomerular filtration and tubular secretion by an anionic transporter system. Given these characteristics, its potential for adverse interactions with other drugs appears limited to those arising from competitive inhibition of excretion by the renal tubular epithelial cell anionic transporter. The terminal plasma elimination half-life is 1.8 h in healthy adults. Renal clearance is inversely related to renal function and averages 23 h after oral administration in individuals with creatinine clearance < 30 ml/min. In vitro studies have demonstrated potent antiviral activity against all strains of influenza A and B tested including avian H5N1 and H9N2 strains recently implicated in human cases in Hong Kong. Studies of both experimental and naturally-occurring influenza in humans have demonstrated efficacy in both the prevention and treatment of influenza A and B infection. The drug is well-tolerated with the only clinically important side effect being mild gastrointestinal upset. Resistance has been uncommonly seen after clinical use; the highest incidence was 5.5% in children treated for influenza A infection for 5 days. Viruses that develop resistance appear to be less virulent in laboratory animals and to replicate less efficiently than parent strains. Although oseltamivir and the M2 ion channel inhibitors, amantadine and rimantadine, have not been directly compared in clinical trials, the greater breadth of spectrum of oseltamivir, its modest side effect profile compared to amantadine and its lesser propensity to engender the emergence of transmissible drug-resistant strains all suggest strongly that oseltamivir is a significant new agent for the prevention and treatment of influenza. A series of controlled trials comparing M2 ion channel inhibitor drugs and the new neuraminidase (NA) inhibitor agents are now needed to test this hypothesis and thereby to further advance the science of antiviral drug use to control influenza.     

Oseltamivir: a clinical and pharmacological perspective

Oseltamivir is the ethyl ester prodrug of the antiviral molecule, oseltamivir carboxylate, a potent and selective inhibitor of influenza A and B neuraminidase (NA) (sialidase). It is highly bioavailable in capsule and suspension formulations and, after conversion to the active metabolite in the liver, distributes throughout the body, including the upper and lower respiratory tract. Oseltamivir carboxylate is 3% bound to human plasma proteins and eliminated through the kidneys by a first-order process as unchanged drug by glomerular filtration and tubular secretion by an anionic transporter system. Given these characteristics, its potential for adverse interactions with other drugs appears limited to those arising from competitive inhibition of excretion by the renal tubular epithelial cell anionic transporter. The terminal plasma elimination half-life is 1.8 h in healthy adults. Renal clearance is inversely related to renal function and averages 23 h after oral administration in individuals with creatinine clearance < 30 ml/min. In vitro studies have demonstrated potent antiviral activity against all strains of influenza A and B tested including avian H5N1 and H9N2 strains recently implicated in human cases in Hong Kong. Studies of both experimental and naturally-occurring influenza in humans have demonstrated efficacy in both the prevention and treatment of influenza A and B infection. The drug is well-tolerated with the only clinically important side effect being mild gastrointestinal upset. Resistance has been uncommonly seen after clinical use; the highest incidence was 5.5% in children treated for influenza A infection for 5 days. Viruses that develop resistance appear to be less virulent in laboratory animals and to replicate less efficiently than parent strains. Although oseltamivir and the M2 ion channel inhibitors, amantadine and rimantadine, have not been directly compared in clinical trials, the greater breadth of spectrum of oseltamivir, its modest side effect profile compared to amantadine and its lesser propensity to engender the emergence of transmissible drug-resistant strains all suggest strongly that oseltamivir is a significant new agent for the prevention and treatment of influenza. A series of controlled trials comparing M2 ion channel inhibitor drugs and the new neuraminidase (NA) inhibitor agents are now needed to test this hypothesis and thereby to further advance the science of antiviral drug use to control influenza.     

Susceptibility of human influenza viruses from Australasia and South East Asia to the neuraminidase inhibitors zanamivir and oseltamivir

Human influenza viruses isolated from Australasia (Australia and New Zealand) and South East Asia were analysed to determine their sensitivity to the NA inhibitor drugs, zanamivir and oseltamivir. A total of 532 strains isolated between 1998 and 2002 were tested using a fluorescence-based assay to measure the relative inhibition of NA activity over a range of drug concentrations. Based on median IC50 values, influenza A viruses (with neuraminidase subtypes N1 and N2) were more sensitive to both the NA inhibitors than were influenza B strains. Influenza A viruses with a N1 subtype and influenza B strains both demonstrated a greater sensitivity to zanamivir than to oseltamivir carboxylate, whereas influenza A strains with a N2 subtype were more susceptible to oseltamivir carboxylate. For each of the neuraminidase types, IC50 values for viruses from Australasia and South East Asia were found to be comparable. Based on the data prior to and following the licensing of the drugs into the respective regions, the use of the NA inhibitors did not appear to have a significant impact on the susceptibility of the viruses tested to zanamivir or oseltamivir carboxylate.     

Characterization of the binding affinities of peramivir and oseltamivir carboxylate to the neuraminidase enzyme

With the continued threat of morbidity and mortality from influenza and the development of resistance to influenza antiviral drugs, there is increasing interest in new treatments, such as the investigational intravenous drug peramivir, and in combination treatments. In this study, we determined the impact of oseltamivir carboxylate on the binding affinity of peramivir/neuraminidase (NA) enzyme complex and vice versa. Influenza NA was incubated with peramivir and oseltamivir carboxylate alone and in combination. Dissociation rates of the enzyme-inhibitor complex measured in the presence of NA substrate for peramivir alone and the combination were similar, suggesting that peramivir competitively inhibits the neuraminidase enzyme and that oseltamivir carboxylate when added to peramivir does not impact the binding affinity of peramivir to the NA enzyme.     

新型甲型H1N1流感病毒神经氨酸酶抑制剂细胞水平评价体系的建立

本文旨在建立以2009新型甲型H1N1流感病毒神经氨酸酶 (neuraminidase, NA) 为靶点的神经氨酸酶抑制剂 (neuraminidase inhibitors, NAIs) 细胞水平评价体系。NA有促进流感病毒释放的作用, 本研究应用重组病毒技术, 通过将表达NA [A/California/04/2009 (H1N1)] 的质粒、表达流感病毒血凝素蛋白HA (hemagglutinin) 的质粒以及表达敲除外壳基因并带有荧光素酶报告基因的HIV-1基因组共转染至病毒生成细胞, 产生以HA、NA为外壳蛋白包裹HIV-1核心的重组病毒。在病毒释放前加入不同浓度的化合物, 收集病毒上清液感染细胞, 通过测定感染率来反映化合物对重组病毒NA的抑制作用。经用阳性对照药物奥司他韦及其羧酸盐证实本模型能够反映化合物对病毒NA的抑制作用; 在此基础上, 本研究还建立了奥司他韦耐药株评价模型。本研究所建立的体系可用于针对新型甲型H1N1流感病毒及其临床耐药株神经氨酸酶抑制剂的寻找和评价。     

New small-molecule drug design strategies for fighting resistant influenza A

Influenza A virus is the major cause of seasonal or pandemic flu worldwide. Two main treatment strategies–vaccination and small molecule anti-influenza drugs are currently available. As an effective vaccine usually takes at least 6 months to develop, anti-influenza small molecule drugs are more effective for the first line of protection against the virus during an epidemic outbreak, especially in the early stage. Two major classes of anti-influenza drugs currently available are admantane-based M2 protein blockers (amantadine and rimantadine) and neuraminidase (NA) inhibitors (oseltamivir, zanamivir, and peramivir). However, the continuous evolvement of influenza A virus and the rapid emergence of resistance to current drugs, particularly to amantadine, rimantadine, and oseltamivir, have raised an urgent need for developing new anti-influenza drugs against resistant forms of influenza A virus. In this review, we first give a brief introduction of the molecular mechanisms behind resistance, and then discuss new strategies in small-molecule drug development to overcome influenza A virus resistance targeting mutant M2 proteins and neuraminidases, and other viral proteins not associated with current drugs.KEY WORDS: Influenza A virus, Drug discovery, Resistance, M2 ion channel, Neuraminidase     

Characterization of a trypsin-dependent avian influenza H5N1-pseudotyped HIV vector system for high throughput screening of inhibitory molecules

In this study, we have generated and characterized an avian influenza H5N1 hemagglutinin (HA), neuraminidase (NA) and M2 ion channel pseudotyped HIV-based vector system (HaNaM-pseudotyped HIV vector). The cleavage site of the HA protein was modified to necessitate trypsin-dependent maturation of the glycoprotein. HA, NA and M2 were efficiently incorporated in HIV vector particles which could transduce different cell lines in a trypsin-dependent manner. Results also showed that the presence of avian influenza M2 and NA proteins maximized both vector production and transduction and that transduction was highly sensitive to the specific NA inhibitor oseltamivir (Tamiflu). H5N1 HaNaM-pseudotyped HIV vector system was also adapted for cell-based high throughput screening of drug candidates against influenza virus infection, and its high sensitivity to the specific oseltamivir validates its potential utility in the identification of new influenza inhibitors. Overall, the trypsin-dependent H5N1-pseudotyped HIV vector can mimic avian influenza virus infection processes with sufficient precision to allow for the identification of new antivirals and to study avian influenza virus biology in a lower biosafety level laboratory environment.     

The 2008-2009 H1N1 influenza virus exhibits reduced susceptibility to antibody inhibition: Implications for the prevalence of oseltamivir resistant variant viruses

A naturally-occurring H275Y oseltamivir resistant variant of influenza A (H1N1) virus emerged in 2007, subsequently becoming prevalent worldwide, via an undetermined mechanism. To understand the antigenic properties of the H275Y variant, oseltamivir resistant and susceptible strains of H1N1 viruses were analyzed by hemagglutination inhibition (HI) and microneutralization assays. HI analysis with H1-positive sera obtained from seasonal flu vaccine immunized and non-immunized individuals, and H1-specific monoclonal antibodies, revealed that resistant strains exhibited a reduced reactivity to these antisera and antibodies in the HI assay, as compared to susceptible strains. Neutralization assay testing demonstrated that oseltamivir resistant H1N1 strains are also less susceptible to antibody inhibition during infection. Mice inoculated with a resistant clinical isolate exhibit 4-fold lower virus-specific antibody titers than mice infected with a susceptible strain under the same conditions. Resistant and sensitive variants of 2009 pandemic H1N1 virus did not exhibit such differences. While HA1 and NA phylogenetic trees show that both oseltamivir resistant and susceptible strains belong to clade 2B, NA D354G and HA A189T substitutions were found exclusively, and universally, in oseltamivir resistant variants. Our results suggest that the reduced susceptibility to antibody inhibition and lesser in vivo immunogenicity of the oseltamivir resistant 2008-2009 H1N1 influenza A virus is conferred by coupled NA and HA mutations, and may contribute to the prevalence of this H1N1 variant.     

In vitro generation and characterisation of an influenza B variant with reduced sensitivity to neuraminidase inhibitors

A contemporary influenza type B virus was passaged in vitro in the presence of increasing concentrations of the neuraminidase inhibitors, zanamivir and oseltamivir carboxylate (0.1-1000 microM over nine passages). After the fifth passage in the presence of zanamivir (10 microM), the virus acquired a Glu 119 Asp neuraminidase mutation (influenza A N2 subtype numbering) in the enzyme active site. After a further three passages, in which growth occurred in 100 microM of zanamivir, a Gln 218 Lys mutation (A (H3) numbering) in the HA1 domain of the haemagglutinin was found. In a fluorescence-based neuraminidase inhibition assay, viruses with the Glu 119 Asp NA mutation had a 32,000-fold reduction in sensitivity to the NA inhibitor zanamivir compared to the wild-type virus, while the mutation resulted in a 105-fold reduction in sensitivity to oseltamivir carboxylate. Viruses grown in the presence of 1000 microM oseltamivir carboxylate did not acquire any neuraminidase mutations but did have a His 103 Gln substitution (A (H3) numbering) in the HA1 region of the haemagglutinin which was demonstrated to significantly reduce receptor binding strength in vitro. Tissue culture assays demonstrated that the HA mutation caused a seven-fold reduction in sensitivity to oseltamivir carboxylate, and a 90-fold reduction in sensitivity to zanamivir.     

Anti-influenza virus activity of peramivir in mice with single intramuscular injection

In the event of an influenza outbreak, antivirals including the neuraminidase (NA) inhibitors, peramivir, oseltamivir, and zanamivir may provide valuable benefit when vaccine production is delayed, limited, or cannot be used. Here we demonstrate the efficacy of a single intramuscular injection of peramivir in the mouse influenza model. Peramivir potently inhibits the neuraminidase enzyme N9 from H1N9 virus in vitro with a 50% inhibitory concentration (IC(50)) of 1.3+/-0.4 nM. On-site dissociation studies indicate that peramivir remains tightly bound to N9 NA (t(1/2)>24h), whereas, zanamivir and oseltamivir carboxylate dissociated rapidly from the enzyme (t(1/2)=1.25 h). A single intramuscular injection of peramivir (10mg/kg) significantly reduces weight loss and mortality in mice infected with influenza A/H1N1, while oseltamivir demonstrates no efficacy by the same treatment regimen. This may be due to tight binding of peramivir to the N1 NA enzymes similar to that observed for N9 enzyme. Additional efficacy studies indicate that a single injection of peramivir (2-20mg/kg) was comparable to a q.d.x 5 day course of orally administered oseltamivir (2-20mg/kg/day) in preventing lethality in H3N2 and H1N1 influenza models. A single intramuscular injection of peramivir may successfully treat influenza infections and provide an alternate option to oseltamivir during an influenza outbreak.     

Current and future antiviral therapy of severe seasonal and avian influenza

The currently circulating H3N2 and H1N1 subtypes of influenza A virus cause a transient, febrile upper respiratory illness in most adults and children ("seasonal influenza"), but infants, the elderly, immunodeficient and chronically ill persons may develop life-threatening primary viral pneumonia or complications such as bacterial pneumonia. By contrast, avian influenza viruses such as the H5N1 virus that recently emerged in Southeast Asia can cause severe disease when transferred from domestic poultry to previously healthy people ("avian influenza"). Most H5N1 patients present with fever, cough and shortness of breath that progress rapidly to adult respiratory distress syndrome. In seasonal influenza, viral replication remains confined to the respiratory tract, but limited studies indicate that H5N1 infections are characterized by systemic viral dissemination, high cytokine levels and multiorgan failure. Gastrointestinal infection and encephalitis also occur. The licensed anti-influenza drugs (the M2 ion channel blockers, amantadine and rimantadine, and the neuraminidase inhibitors, oseltamivir and zanamivir) are beneficial for uncomplicated seasonal influenza, but appropriate dosing regimens for severe seasonal or H5N1 viral infections have not been defined. Treatment options may be limited by the rapid emergence of drug-resistant viruses. Ribavirin has also been used to a limited extent to treat influenza. This article reviews licensed drugs and treatments under development, including high-dose oseltamivir; parenterally administered neuraminidase inhibitors, peramivir and zanamivir; dimeric forms of zanamivir; the RNA polymerase inhibitor T-705; a ribavirin prodrug, viramidine; polyvalent and monoclonal antibodies; and combination therapies.     

Biological evaluation of anti-influenza viral activity of semi-synthetic catechin derivatives

Catechin derivatives with different alkyl chain length and aromatic ring substitutions at the 3-hydroxyl group were synthesized from epigallocatechin (EGC) and (+)-catechin (C) and their anti-influenza viral activity were evaluated in vitro and in ovo. Pronounced antiviral activity was observed for derivatives carrying moderate chain length (7-9 carbons) as compared to those with aromatic rings, whereas the 5'-hydroxyl group of the trihydroxy benzyl moiety did not significantly contribute to antiviral activity. The derivatives exerted inhibitory effects for all six influenza subtypes tested including three major types of currently circulating human influenza viruses (A/H1N1, A/H3N2 and B type), H2N2 and H9N2 avian influenza virus. The compounds strongly inhibited adsorption of the viruses on red blood cell (RBC). They also restricted the growth of avian influenza virus in ovo with minimum inhibition concentration (MIC) of 5-10 microM far exceeding the neuraminidase (NA) inhibitor oseltamivir or M2 proton channel inhibitor amantadine. The antiviral activity appears to be mediated by interaction with hemagglutinin (HA)/viral membrane rendering HA less fusogenic at the initial stage of infection. The broad spectrum activity against various subtypes of influenza viruses may complement the limitations of current antivirals and contribute for managing potentially emerging influenza pandemic. The structure-activity data of catechin derivatives may usefully guideline future research endeavors for applying green tea catechins as alternative anti-viral agents.     

Arylazolyl(azinyl)thioacetanilide. Part 9: Synthesis and biological investigation of thiazolylthioacetamides derivatives as a novel class of potential antiviral agents

In continuation of our endeavor to develop new, potent, selective and less toxic antiviral agents, a novel series of 2-(2-amino/chloro-4-(2,4-dibromophenyl) thiazol-5-ylthio)acetamide derivatives was synthesized via an expeditious route and evaluated for their anti-HIV activities against wild-type virus and clinically relevant mutant strains, and for their anti-influenza virus activities against influenza A (H1N1 and H3N2) and influenza B in cellular assays. The selected active compounds were also assayed for their enzymic inhibitory activities. The results showed that some 2-chloro substituted thiazolylthioacetamide derivatives possessed potent activity against wild type HIV-1 and several key mutant strains (E138K, K103N, L100I) of HIV-1 in MT-4 cells with EC(50) values in micromolar range. Two 2-amino substituted thiazole derivatives 8a7 and 8a8 displayed significant potency against influenza A/H1N1 in MDCK cells with EC(50) values much lower than that of oseltamivir carboxylate, ribavirin, amantadine and rimantadine. Though the mechanism of actions is still unclear, these novel thiazolylthioacetamides might serve as original leads for further pharmacological investigations as potential therapeutic agents against HIV-1 or influenza virus.     

Genotypic and phenotypic resistance of pandemic A/H1N1 influenza viruses circulating in Germany

In response to the rapid global spread of an antigenically novel A/H1N1 influenza virus in 2009, the World Heath Organization (WHO) recommended surveillance and monitoring for antiviral resistance of influenza viruses. We designed and evaluated pyrosequencing (PSQ)-based genotypic assays for high-throughput analysis of the susceptibility of pandemic A/H1N1 influenza viruses to neuraminidase (NA) inhibitors. A total of 1570 samples circulating in Germany between April 2009 and April 2010 were tested for determination of molecular markers of resistance to the NA inhibitors oseltamivir and zanamivir, and 635 of them were evaluated by phenotypic fluorescence-based assay with MUNANA substrate. Eight (0.5%) viruses were resistant to oseltamivir due to the H274Y NA substitution (N2 numbering). Six of these oseltamivir-resistant cases were treatment-related; four of them were selected in immunocompromised patients, two in patients suffered from chronic diseases. The two remaining oseltamivir-resistant viruses seem to have evolved in the absence of drug treatment and were isolated from immunocompetent healthy patients. All tested A/H1N1 pandemic viruses were sensitive to zanamivir. In addition, analysis of 1011 pandemic A/H1N1 virus samples by a PSQ-based assay according to the WHO protocol revealed the presence of mutation S31N in the M2 protein that conferred resistance to M2 ion channel inhibitors. Our data demonstrate a low incidence of oseltamivir-resistant pandemic A/H1N1 influenza variants isolated under drug selection pressure as well as community-acquired or naturally evolving viruses.     

Analysis of influenza viruses from patients clinically suspected of infection with an oseltamivir resistant virus during the 2009 pandemic in the United States

During the 2009 influenza pandemic, the Centers for Disease Control and Prevention provided antiviral susceptibility testing for patients infected with suspected drug-resistant viruses. Specimens from 72 patients admitted to an intensive care unit or with a severe immunocompromising condition, who failed to clinically improve after oseltamivir treatment, were accepted for testing. Respiratory specimens were tested for the presence of the oseltamivir resistance-conferring H275Y substitution in the neuraminidase (NA) by pyrosequencing. Virus isolates propagated in MDCK cells were tested in phenotypic NA inhibition (NI) assays using licensed NA inhibitors (NAIs), zanamivir and oseltamivir, and investigational NAIs, peramivir and laninamivir. Conventional sequencing and plaque purification were conducted on a subset of viruses. Pyrosequencing data were obtained for 87 specimens collected from 58 of the 72 (81%) patients. Of all patients, 27 (38%) had at least one specimen in which H275Y was detected. Analysis of sequential samples from nine patients revealed intra-treatment emergence of H275Y variant and a shift from wildtype-to-H275Y in quasispecies during oseltamivir therapy. A shift in the H275Y proportion was observed as a result of virus propagation in MDCK cells. Overall, the NI method was less sensitive than pyrosequencing in detecting the presence of H275Y variants in virus isolates. Using the NI method, isolates containing H275Y variant at ? 50% exhibited resistance to oseltamivir and peramivir, but retained full susceptibility to zanamivir. H275Y viruses recovered from two patients had an additional substitution I223K or I223R that conferred a 38–52- and 33–97-fold enhancement in oseltamivir- and peramivir-resistance, respectively. These viruses also showed decreased susceptibility to zanamivir and laninamivir. These data suggest that pyrosequencing is a powerful tool for timely detection of NAI resistant viruses and that NI assays are needed for comprehensive testing to detect novel resistance substitutions.     

Susceptibility of avian influenza viruses of the N6 subtype to the neuraminidase inhibitor oseltamivir

Avian influenza viruses are a source of genetic material that can be transmitted to humans through direct introduction or reassortment. Although there is a wealth of information concerning global monitoring for antiviral resistance among human viruses of the N1 and N2 neuraminidase (NA) subtypes, information concerning avian viruses of these and other NA subtypes is limited. We undertook a surveillance study to investigate the antiviral susceptibility of avian influenza N6 NA viruses, the predominant subtype among wild waterfowl. We evaluated 73 viruses from North American ducks and shorebirds for susceptibility to the NA inhibitor oseltamivir in a fluorescence-based NA enzyme inhibition assay. Most (90%) had mean IC₅₀ values ranging from <0.01 to 5.0 nM; 10% were from 5.1 to 50.0 nM; and none were >50.0 nM. Susceptibility to oseltamivir remained stable among all isolates collected over approximately three decades (P ? 0.74). Two isolates with I222V NA substitution had moderately reduced susceptibility to oseltamivir in vitro (IC₅₀, 30.0 and 40.0 nM). One field sample was a mixed population containing an avian paramyxovirus (APMV) and H4N6 influenza virus, as revealed by electron microscopy and hemagglutination inhibition assays with a panel of anti-APMV antisera. This highlights the importance of awareness and careful examination of non-influenza pathogens in field samples from avian sources. This study showed that oseltamivir-resistant N6 NA avian influenza viruses are rare, and must be tested both phenotypically and genotypically to confirm resistance.     

Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes

Since 2003, highly pathogenic A(H5N1) influenza viruses have been the cause of large-scale death in poultry and the subsequent infection and death of over 140 humans. A group of 55 influenza A(H5N1) viruses isolated from various regions of South East Asia between 2004 and 2006 were tested for their susceptibility to the anti-influenza drugs the neuraminidase inhibitors and adamantanes. The majority of strains were found to be fully sensitive to the neuraminidase inhibitors oseltamivir carboxylate, zanamivir and peramivir; however two strains demonstrated increased IC50 values. Sequence analysis of these strains revealed mutations in the normally highly conserved residues 116 and 117 of the N1 neuraminidase. Sequence analysis of the M2 gene showed that all of the A(H5N1) viruses from Vietnam, Malaysia and Cambodia contained mutations (L26I and S31N) associated with resistance to the adamantane drugs (rimantadine and amantadine), while strains from Indonesia were found to be a mix of both adamantane resistant (S31N) and sensitive viruses. None of the A(H5N1) viruses from Myanmar contained mutations known to confer adamantane resistance. These results support the use of neuraminidase inhibitors as the most appropriate class of antiviral drug to prevent or treat human A(H5N1) virus infections.