深圳市2008-2009年A型H1N1季节性流感病毒神经氨酸酶抑制剂耐药性监测

目的 对深圳市2008-2009年分离到的H1N1季节性流感病毒神经氨酸酶(NA)抑制剂的耐药性进行监测.方法 根据原始临床样本的采集时间,按周抽取了55株2008-2009年分离到的H1N1季节性流感病毒,对其NA片段进行全长测序,选取WHO推荐的疫苗株和部分国内外分离到的H1N1季节性流感病毒作为参考株,运用Mega3.1软件进行种系发生树的构建、耐药相关位点及糖基化位点的分析.结果 对NA片段的序列分析发现2008年有2株(7.1%)出现了H275Y突变,但是2009年则有25株(92.6%)出现了该突变.提示H275Y达菲耐药突变株成为了2009年深圳市社区传播的优势株.同时还发现了一株Q136K变异株,显示对乐感清出现耐药.分子进化分析结果显示,H275Y变异成为了毒株在系统进化树上分布的主要依据.所有的深圳株NA片段上潜在的糖基化位点序列保守.结论 大量H275Y达菲耐药株的出现提示在今后的工作中应当密切关注流感病毒的耐药进展,进一步加强其耐药机制的研究.

Abstract：

Objective To analyze neuraminidase(NA) inhibitor resistance of seasonal H1N1 influenza A viruses isolated in Shenzhen during 2008 to 2009. Methods The NA gene of these viruses were sequenced. Phylogenetic analysis of the sequences was performed with Mega3. 1 software. Results In 2008, most isolates of the seasonal H1 N1 virus were susceptible to neuraminidase inhibitors, but the H275Y mutation in the neuraminidase gene region associated with high-level oseltamivir resistance had been detected in 92.6% of the strains isolated in 2009. Furthermore, a strain with Q136K was found, which showed the resistance to Zanamivir. Conclusion In the light of emerging resistance, close monitoring and understanding of the nature and dynamics of resistance mutations in influenza virus should be a priority.     

Emergence of an Oseltamivir-Resistant Influenza A/H3N2 Virus in an Elderly Patient Receiving a Suboptimal Dose of Antiviral Prophylaxis

We report the emergence of an influenza virus A/H3N2-E119V neuraminidase variant from an elderly patient with renal dysfunction who received a suboptimal dose of oseltamivir prophylaxis. In neuraminidase inhibition assays, the E119V variant showed a 413-fold increase in the 50% inhibitory oseltamivir concentration and grew at titers comparable to those of the wild type in vitro.     

Influenza virus A(H1N1)pdm09 hemagglutinin polymorphism and associated disease in southern Germany during the 2010/11 influenza season

A novel influenza A virus emerged in early 2009 to cause the first influenza pandemic of the 21^st century. Understanding the evolution of influenza virus is crucial to determine pathogenesis, vaccine efficacy, and resistance to antiviral drugs. In this study, we investigated the molecular evolution of influenza virus A(H1N1)pdm09 in the 2010/11 influenza season in southern Germany by sequence analysis of the influenza virus hemagglutinin gene from 25 patients with mild, moderate, and severe disease. Phylogenetic analysis revealed co-circulation of different genetic groups. The D222G mutation, which had previously been observed in severe cases, was not detected. Immunocompromised patients were not affected more severely than non-immunocompromised patients (p>0.05), although longer shedding was observed in some of them. Interestingly, additional mutations and potential glycosylation sites were detected in samples from the lower respiratory tract in two patients, but not in the corresponding upper respiratory tract specimens. The H275Y mutation in the influenza virus neuraminidase gene, known to confer resistance to the neuraminidase inhibitor oseltamivir, was detected in one patient.     

Ultrasensitive detection of drug-resistant pandemic 2009 (H1N1) influenza A virus by rare-variant-sensitive high-resolution melting-curve analysis

Oseltamivir (Tamiflu), an oral neuraminidase inhibitor, has been widely used to treat pandemic 2009 (H1N1) influenza A. Although a majority of 2009 (H1N1) influenza A virus remains oseltamivir susceptible, the threat of resistance due to the His275Tyr mutation is highlighted by the limitations of alternative therapies and the potential for rapid, global fixation of this mutation in the circulating influenza A virus population. In order to better understand the emergence of resistance, we developed a rare-variant-sensitive high-resolution melting-curve analysis method (RVS-HRM) that is able to detect the His275Tyr oseltamivir resistance mutation to 0.5% in a background of susceptible virus. We applied RVS-HRM to clinical specimens from patients who developed oseltamivir resistance and demonstrated the ultrasensitive detection of influenza A virus N1 neuraminidase quasispecies. Interestingly, we were unable to detect the oseltamivir resistance mutation in pretreatment samples, suggesting that resistant virus does not reach even this very low detection threshold until exposed to selective drug pressure. Thus, patients naive to oseltamivir are most likely to be susceptible when this drug is used as a first-line treatment modality.     

Characterization of oseltamivir‐resistant influenza A(H1N1)pdm09 viruses in Taiwan in 2009–2011

The early isolated swine‐origin influenza A(H1N1)pdm09 viruses were susceptible to oseltamivir; however, there is a concern about whether oseltamivir‐resistant influenza A(H1N1)pdm09 viruses will spread worldwide as did the oseltamivir‐resistant seasonal influenza A(H1N1) viruses in 2007–2008. In this study, the frequency of oseltamivir resistance in influenza A(H1N1)pdm09 viruses was determined in Taiwan. From May 2009 to April 2011, 1,335 A(H1N1)pdm09‐positive cases in Taiwan were tested for the H275Y mutation in the neuraminidase (NA) gene that confers resistance to oseltamivir. Among these, 15 patients (1.1%) were found to be infected with H275Y virus. All the resistant viruses were detected after the patients have received the oseltamivir. The overall monthly ratio of H275Y‐harboring viruses ranged between 0% and 2.88%, and the peak was correlated with influenza epidemics. The genetic analysis revealed that the oseltamivir‐resistant A(H1N1)pdm09 viruses can emerged from different variants with a great diversity under drug pressure. The ratio of NA/HA activities in different clades of oseltamivir‐resistant viruses was reduced compared to those in the wild‐type viruses, indicating that the balance of NA/HA in the current oseltamivir‐resistant influenza A(H1N1)pdm09 viruses was interfered. It is possible that H275Y‐bearing A(H1N1)pdm09 virus has not yet spread globally because it lacks the essential permissive mutations that can compensate for the negative impact on fitness by the H275Y amino acid substitution in NA. Continuous monitoring the evolution patterns of sensitive and resistant viruses is required to respond to possible emergence of resistant viruses with permissive genetic background which enable the wide spread of resistance. J. Med. Virol. 85:379–387, 2013. © 2012 Wiley Periodicals, Inc.     

First isolation of an H1N1 avian influenza virus from wild terrestrial non-migratory birds in Argentina

A type A avian influenza (AI) virus was isolated from dead or severely ill red-winged tinamous (Rhynchotus rufescens) found in a hunting ground in April 2008 in Argentina. The subtype of A/red-winged tinamou/Argentina/MP1/2008 was determined as H1N1 by sequence analysis. The cleavage site of the viral hemagglutinin corresponded to a low pathogenic influenza virus, although the clinical presentation and pathological studies suggest that the virus was pathogenic for red-winged tinamous. Phylogenetic analysis of the viral genome suggested that while the hemagglutinin and neuraminidase genes were related to AIV from North America, the internal genes were most closely related to other South American isolates. These findings support the postulated South American phylogenetic lineage for AIV PB2, PB1, PA, M and NS genes, and suggest that the evolutionary pathways of HA and NA genes involve exchanges between the Northern and Southern hemispheres.     

In vitro selection of influenza B viruses with reduced sensitivity to neuraminidase inhibitors

We and others have previously isolated influenza B viruses with reduced sensitivity to neuraminidase (NA) inhibitors (oseltamivir and zanamivir) from patients who were never exposed to these drugs. It was unclear whether the NA substitutions found in these influenza B isolates arose spontaneously or were caused by selective pressure. Here, we obtained influenza B viruses with reduced NA inhibitor sensitivity by in vitro selection with NA inhibitors. We found that these viruses possessed the same NA substitutions as those previously found in viruses isolated from untreated patients. These results suggest that these NA substitutions were selected in patients who were treated with an NA inhibitor and that the resistant variants were then transmitted to others.     

H1N1V influenza epidemic of 2009 in Russia

The paper describes dynamics, distribution and morbidity rate during the 2009 A(H1N1)v influenza epidemic in Russia. The epidemic appears to have been especially severe in the cities of the Far-East and Siberian Federal Districts where the average morbidity rate ranged from 6.4% to 19.2% (mean 10.3%) and the epidemic duration from 7.8 to 8 weeks. In less affected Southern and Central Federal Districts A(H1N1)v influenza occurred in 5.7% of the population. Schoolchildren aged 7-4 years showed the highest morbidity rate of 28.8%. The age group of 18-53 years accounted for 79.4% of the total lethality. Viral isolates were genetically stable and exhibited 98.9% hemagglutnin (HA) homology with reference viruses. None of the strains had an amino acid substitution at position 275 of neuraminidase (NA) responsible for resistance to oseltamivir. Towards the end of the epidemic, the viral population displayed a significant rise in the number of strains containing mutations in 4 genes (4 HA, 2 NA, 2 PB2 and 1 PA mutations respectively). 26.7% of the viral isolates obtained in the end of the epidemic had D222G substitution responsible for tropism of viruses to lung tissues. Epidemiologically, the 2009 A(H1NI)v influenza epidemic is described as moderate based on the absence of pathogenicity determinants typical of both A(H1N1) influenza virus of 1918 and A(H5N1) virus. The paper compares the 2009 epidemic with those caused by A/Honkong/68 and A/USSR/ 90/77 viruses. The necessity of classification for the discrimination between A(H1N1) subtype viruses is emphasized.     

Performance of rapid-test kits for the detection of the pandemic influenza A/H1N1 virus

The early detection of pandemic influenza strains is a key factor for clinicians in treatment decisions and infection control practices. The aims of this study were to determine the analytical sensitivity and clinical performance of the commercially available influenza rapid tests in Taiwan. Four rapid tests for influenza virus (BinaxNow test, QuickVue test, TRU test, and Formosa Rapid test) were evaluated for their detection limit against four influenza viruses (the 2009 pandemic influenza A virus H1N1, seasonal influenza virus H1N1, H3N2, and influenza B virus) circulating in Taiwan. The viral load of these isolates were quantified by rtRT-PCR and then diluted 2-fold serially for the comparison. The lowest detectable viral load of the pandemic influenza A virus H1N1 by the Formosa Rapid test, QuickVue test, TRU test, and Binax Now test was 5.3 × 10⁴, 1.0 × 10⁵, 1.0 × 10⁵, and 4.2 × 10⁵ copies/μL, respectively. Of these four tests, the two most sensitive tests (the QuickVue test and the Formosa Rapid test) were chosen to evaluate 62 nasopharyngeal specimens from patients who were suspected of infection with pandemic influenza A virus H1N1. The positive rate for the Formosa Rapid test and the QuickVue test were 53.2% (33/62) and 45.2% (28/62) (McNemar's test, P = 0.125), respectively. In conclusion, the Formosa Rapid test was the most sensitive test in the present study for the detection of influenza antigens and its clinical performance was similar to that of the QuickVue test (Kappa = 0.776). This suggests that the Formosa Rapid test could be used to aid clinical decision making in primary health care settings during outbreaks of influenza.     

In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity

Acquisition of α2-6 sialoside receptor specificity by α2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding α2-6 sialosides, we identified four variant viruses with amino acid substitutions in the hemagglutinin (S227N, D187G, E190G, and Q196R) that revealed modestly increased α2-6 and minimally decreased α2-3 binding by glycan array analysis. However, a mutant virus combining Q196R with mutations from previous pandemic viruses (Q226L and G228S) revealed predominantly α2-6 binding. Unlike the wild type H5N1, this mutant virus was transmitted by direct contact in the ferret model although not by airborne respiratory droplets. However, a reassortant virus with the mutant hemagglutinin, a human N2 neuraminidase and internal genes from an H5N1 virus was partially transmitted via respiratory droplets. The complex changes required for airborne transmissibility in ferrets suggest that extensive evolution is needed for H5N1 transmissibility in humans.     

Characterization of human H1N1 influenza virus variants selected in vitro with zanamivir in the presence of sialic acid-containing molecules

Understanding the molecular mechanisms of influenza virus resistance to neuraminidase inhibitors is a main concern for their clinical use. In an attempt to reproduce in vivo selective conditions where influenza virus resistance to neuraminidase inhibitors can occur the zanamivir selection of an A/H1N1 influenza virus strain was carried out in Madin-Darby canine kidney cells performed in the presence or absence of sialic acid-containing inhibitor analogues that act as virus decoy receptors. The zanamivir-selected variants passaged in the presence of sialic acid-containing molecules resembling the human-like virus receptor lost the ability to bind red blood cells. Furthermore, whereas all zanamivir-selected variants exhibited a robust reduction in susceptibility to zanamivir in plaque assays only those obtained after extensive passages acquired a powerful neuraminidase enzyme resistance to zanamivir and oseltamivir. Evidence that balanced neuraminidase and hemagglutinin activities mediated by mutations induced during selection could play a role in the decrease of virus replication susceptibility to zanamivir is reported.     

Neuraminidase inhibitor susceptibility of swine influenza A viruses isolated in Germany between 1981 and 2008

European swine influenza A viruses donated the matrix protein 2 as well as the neuraminidase (NA) gene to pandemic influenza A (H1N1) viruses that emerged in 2009. As a result, the latter became amantadine resistant and neuraminidase inhibitor (NAI) susceptible. These recent developments reflecting the close connection between influenza A virus infection chains in humans and pigs urge an antiviral surveillance within swine influenza A viruses. Here, NAI susceptibility of 204 serologically typed swine influenza A viruses of subtypes H1N1, H1N2, and H3N2 circulating in Germany between 1981 and 2008 was analyzed in chemiluminescence-based NA inhibition assays. Mean 50% inhibitory concentrations of oseltamivir and zanamivir indicate a good drug susceptibility of tested viruses. As found for human isolates, the oseltamivir and zanamivir susceptibility was subtype-specific. So, swine influenza A (H1N1) viruses were just as susceptible to oseltamivir as to zanamivir. In contrast, swine H1N2 and H3N2 influenza A viruses were more sensitive to oseltamivir than to zanamivir. Furthermore, reduction in plaque size and virus spread by both drugs was tested with selected H1N1 and H1N2 isolates in MDCK cells expressing similar amounts of α2.3- and α2.6-linked sialic acid receptors. Data obtained in cell culture-based assays for H1N1 isolates correlated with that from enzyme inhibition assays. But, H1N2 isolates that are additionally glycosylated at Asn158 and Asn163 near the receptor-binding site of hemagglutinin (HA) were resistant to both NAI in MDCK cells. Possibly, these additional HA glycosylations cause a misbalance between HA and NA function that hampers or abolishes NAI activity in cells.     

Antiviral activity of baicalin against influenza A (H1N1/H3N2) virus in cell culture and in mice and its inhibition of neuraminidase

Scutellaria baicalensis Georgi, a Chinese herbal decoction, has been used for the treatment of the common cold, fever and influenza virus infections. In previous studies, we found that oral administration of baicalein resulted in the inhibition of influenza A virus replication in vivo, which was linked to baicalin in serum. However, the effective dose and underlying mechanisms of the efficacy of baicalin against influenza A virus have not been fully elucidated. In this study, the antiviral effects of baicalin in influenza-virus-infected MDCK cells and mice were examined. The neuraminidase inhibition assay was performed to investigate the mechanism of action of baicalin. In vitro results showed that baicalin exhibited a half-maximal effective concentration (EC₅₀) of 43.3 μg/ml against the influenza A/FM1/1/47 (H1N1) virus and 104.9 μg/ml against the influenza A/Beijing/32/92 (H3N2) virus. When added to MDCK cell cultures after inoculation with influenza virus, baicalin demonstrated obvious antiviral activity that increased in a dose-dependent manner, indicating that baicalin affected virus budding. Baicalin had clear inhibitory effects against neuraminidases, with half-maximal inhibitory concentration (IC₅₀) of 52.3 μg/ml against the influenza A/FM1/1/47 (H1N1) virus and 85.8 μg/ml against the influenza A/Beijing/32/92 (H3N2) virus. In vivo studies showed that an intravenous injection of baicalin effectively reduced the death rate, prolonged the mean day to death (MDD) and improved the lung parameters of mice infected with influenza A virus. These results demonstrate that baicalin acts as a neuraminidase inhibitor, with clear inhibitory activities that are effective against different strains of influenza A virus in both cell culture and a mouse model, and that baicalin has potential utility in the management of influenza virus infections.     

Dynamics of clinical symptoms in patients with pandemic influenza A (H1N1)

We evaluated the dynamics of clinical symptoms of 2009 pandemic influenza A (H1N1) using a four-point scale sheet. The most frequent symptoms were fever and cough. The sum of symptom score was high during the first 4 days. Systemic symptoms peaked earlier, by day 2, and resolved faster than upper respiratory symptoms and lower respiratory symptoms after oseltamivir treatment. The lower respiratory symptoms resolved slowly over 2 weeks. The 2009 pandemic influenza A (H1N1) virus might involve primarily lower respiratory tract and could be the main cause of pneumonia.     

GENEDIA Multi Influenza Ag Rapid Test for detection and H1, H3, and H5 subtyping of influenza viruses

BackgroundRapid identification and subtype determination of influenza virus is important in managing infected patients. Rapid influenza diagnostic tests (RIDTs) are widely used in this manner, but most can only detect influenza A and B viruses without subtyping. A new RIDT, GENEDIA Multi Influenza Ag Rapid Test (GENEDIA), was developed for detection of influenza A and B viruses and also subtyping of influenza A to H1, H3, H5 which has not been possible with other RIDTs.ObjectivesAssess the performance of GENEDIA.Study designNasopharyngeal swabs were collected from 274 clinically suspected patients (influenza A/H1N1/2009 (n = 50), influenza A/H3 (n = 50), influenza B (n = 73) and influenza-negative (n = 101)) and analyzed with the real-time RT-PCR, GENEDIA, SD Bioline Influenza Ag, and Alere BinaxNow Influenza A&B Card. Also, 46 fecal specimens (H5N2 (n = 3), H5N3 (n = 3)) of spot-billed duck were analyzed with RT-PCR and GENEDIA.ResultsCompared to real-time RT-PCR, the sensitivities of GENEDIA, SD Bioline Influenza Ag, and Alere BinaxNow Influenza A&B Card were 73.0%, 57.0%, 58.0% for influenza A, respectively, and 68.5%, 65.8%, 57.5% for influenza B, respectively. Specifically, the sensitivity of GENEDIA was 70.0% for influenza A/H1N1/2009 and 76.0% for influenza A/H3. From the avian influenza samples, GENEDIA detected all six H5 subtype without any cross-reactions.ConclusionThe GENEDIA Multi Influenza Ag Rapid Test was sensitive in detecting influenza viruses compared with other commercial RIDTs and also useful for rapid subtype determination of influenza A.     

Identification of Novel Compounds against an R294K Substitution of Influenza A (H7N9) Virus Using Ensemble Based Drug Virtual Screening

Influenza virus H7N9 foremost emerged in China in 2013 and killed hundreds of people in Asia since they possessed all mutations that enable them to resist to all existing influenza drugs, resulting in high mortality to human. In the effort to identify novel inhibitors combat resistant strains of influenza virus H7N9; we performed virtual screening targeting the Neuraminidase (NA) protein against natural compounds of traditional Chinese medicine database (TCM) and ZINC natural products. Compounds expressed high binding affinity to the target protein was then evaluated for molecular properties to determine drug-like molecules. 4 compounds showed their binding energy less than -11Kcal/mol were selected for molecular dynamics (MD) simulation to capture intermolecular interactions of ligand-protein complexes. The molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method was utilized to estimate binding free energy of the complex. In term of stability, NA-7181 (IUPAC namely {9-Hydroxy-10-[3-(trifluoromrthyl) cyclohexyl]-4.8-diazatricyclo [6.4.0.02,6]dodec-4-yl}(perhydro-1H-inden-5-yl)formaldehyde) achieved stable conformation after 20ns and 27ns for ligand and protein root mean square deviation, respectively. In term of binding free energy, 7181 gave the negative value of -30.031 (KJ/mol) indicating the compound obtained a favourable state in the active site of the protein.     

Influenza Antiviral Resistance Testing in New York and Wisconsin, 2006 to 2008: Methodology and Surveillance Data

The need for effective influenza antiviral susceptibility surveillance methods has increased due to the emergence of near-universal adamantane resistance in influenza A/H3N2 viruses during the 2005-2006 season and the appearance of oseltamivir resistance in the influenza A/H1N1 virus subtype during the 2007-2008 season. The two classes of influenza antivirals, the neuraminidase inhibitors (NAIs) and the adamantanes, are well characterized, as are many mutations that can confer resistance to these drugs. Adamantane resistance is imparted mainly by a S31N mutation in the matrix gene, while NAI resistance can result from a number of mutations in the neuraminidase gene. During the 2007-2008 season, a neuraminidase mutation (H274Y) conferring resistance to the NAI oseltamivir emerged worldwide in the A/H1N1 virus subtype. Surveillance methodology and data from New York (NY) and Wisconsin (WI) for the 2006-2007 and 2007-2008 influenza seasons are presented. We used an existing pyrosequencing method (R. A. Bright et al., Lancet 366:1175-1181, 2005) and a modified version of this method for detection of adamantane resistance mutations. For NAI resistance mutation detection, we used a mutation-specific pyrosequencing technique and developed a neuraminidase gene dideoxy sequencing method. Adamantane resistance in the A/H3N2 virus samples was 100% for 2007-2008, similar to the 99.8% resistance nationwide as reported by the CDC. Adamantane resistance was found in only 1.2% of NY and WI A/H1N1 virus samples, compared to that found in 10.8% of samples tested nationwide as reported by the CDC. Influenza A/H1N1 virus H274Y mutants were found in 11.1% of NY samples for 2007-2008, a level comparable to the 10.9% nationwide level reported by the CDC; in contrast, mutants were found in 17.4% of WI samples. These results indicate the need for regional influenza antiviral surveillance.Influenza virus is a highly contagious agent that can cause symptoms ranging from mild discomfort to severe respiratory disease and is a contributing factor in over 30,000 deaths annually in the United States alone (27). Currently, the major health-care strategies for controlling the spread of influenza entail two main approaches, vaccination and the use of antiviral drugs (21). Vaccines are the most effective method of limiting the spread of influenza and preventing symptoms (15). However, vaccine efficacy is limited to the strains selected for each year''s vaccine. Since vaccine development and production typically require several months, prediction of the major seasonal strains can be difficult and is occasionally inaccurate. Also, vaccination compliance can vary from year to year, and outbreaks in long-term-care facilities can occur regardless of the residents'' vaccination statuses. Thus, antiviral drugs may serve as the initial agents for the prevention and treatment of influenza. Currently, two classes of influenza antiviral drugs are approved for influenza prophylaxis and treatment in the United States: the M2 ion channel blockers (including amantadine and rimantadine, collectively referred to as the adamantanes) and the neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir).Adamantanes are effective only against influenza type A virus; they function by blocking ion flow through the 97-amino-acid viral proton channel (the M2 protein of the matrix gene), which is necessary for replication (1). Adamantanes have been shown to be effective agents for the treatment of influenza symptoms as well as for prophylaxis (13, 23). However, recent publications have documented a rapid increase in the incidence of adamantane-resistant influenza virus strains during the 2005-2006 influenza season (4, 12). Adamantane resistance is associated with mutations in a short region of the M2 protein sequence encompassed in nucleotides 789 to 815 of the matrix gene. Thus far, five resistance mutations, in the codons for amino acids 26, 27, 30, 31, and 34, have been reported. The predominant circulating resistance mutation reported affects amino acid 31, resulting in a serine-to-asparagine (S31N) change (24, 26). During the 2005-2006 influenza season, the Centers for Disease Control and Prevention (CDC) found that more than 90% of A/H3N2 viruses circulating in the United States had the S31N adamantane resistance mutation and recommended that adamantanes not be used for treatment or prophylaxis of influenza until susceptibility can be reestablished (5). The results also indicated that only 4% of A/H1N1 viruses tested were adamantane resistant (12). In Asia during the same period, the levels of resistance were slightly higher: nearly all A/H3N2 and 15% of A/H1N1 viruses tested had an adamantane resistance mutation (12).The use of NAIs has increased as a result of the decreased efficacy of adamantanes against such a large proportion of circulating influenza viruses. NAI antivirals are analogues of sialic acid, the biological neuraminidase (NA) substrate. The NAIs disrupt viral replication by occupying the NA active site, thus limiting the binding of sialic acid and the subsequent NA cleavage of host cell receptors (21, 28). Although currently not as common as adamantane resistance, NAI resistance can develop as a result of amino acid-altering mutations in the NA gene of influenza virus. Characterized NAI resistance mutations, distributed throughout the NA gene, result in catalytic or structural changes that affect the sialic acid binding site. Several studies have developed models predicting that the increased use of NAI antivirals, such as during an influenza pandemic, may result in an increase in circulating NAI-resistant viruses, which could greatly decrease the overall efficacy of antivirals used in a pandemic situation (10, 20). In Japan, oseltamivir is often prescribed for prophylactic therapy, as well as for treatment of influenza cases. The widespread use of the drug has been cited as a reason for higher rates of resistance in Japan, previously found to be as high as 18% in pediatric cases (18). In the early part of the 2007-2008 influenza season, an oseltamivir-resistant A/H1N1 virus strain was reported first in Norway and subsequently in other countries worldwide (19, 29). In many European nations where oseltamivir use is very limited, a high percentage of the 2007-2008 influenza season''s A/H1N1 virus strains had the oseltamivir resistance mutation, H274Y. This finding may indicate that oseltamivir resistance can develop and spread, even in the absence of the selective pressure exerted by excessive use of the drug. Despite variable reports on the transmissibility of drug-resistant influenza viruses (3, 16), the H274Y mutant spread rapidly throughout Europe during the 2007-2008 season. Thus far, the oseltamivir-resistant virus has remained susceptible to zanamivir (7, 22).Clearly, timely and reliable methods are needed to monitor the development of resistance to influenza antivirals if these drugs are to be used effectively in both seasonal and pandemic situations. Here, we describe such methods for monitoring the mutations that are known to increase resistance to either adamantanes or NAIs. Pyrosequencing allows the rapid screening of the critical region of the influenza A virus matrix gene, for detection of specific adamantane resistance mutations, or of the NA gene, for detection of a specific NA resistance mutation (i.e., H274Y). For more-extensive NAI resistance genotypic analysis, a method that uses traditional Sanger sequencing and that is based on newly compiled databases of influenza virus sequences is described. We also report results, acquired with these methods, from surveillance programs conducted by two state public health laboratories during the 2006-2007 and 2007-2008 influenza seasons in New York (NY) and Wisconsin (WI).