Impact of the H274Y Substitution on N1, N4, N5, and N8 Neuraminidase Enzymatic Properties and Expression in Reverse Genetic Influenza A Viruses.

The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.

Optimization of an allelic discrimination real-time RT-PCR assay for detection of H275Y oseltamivir resistance gene mutation among influenza A(H1N1)pdm09 patients from 2020 to 2022.

Influenza virus is known to cause mild to severe respiratory infections and is also prone to genetic mutations. Of all the mutations, neuraminidase (NA) gene mutations are a matter of concern, as most approved antivirals target this protein. During the 2020 influenza season, an emergence of mutation in the NA gene, affecting the binding of the World Health Organization (WHO)-recommended probes to the specific site of the NA gene, was reported by our group. As a result of this mutation, the WHO-recommended allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was unable to detect wild-type (H275) or mutant oseltamivir-resistant (Y275) strains of influenza A(H1N1)pmd09 viruses. In the current study, the WHO-recommended probes were redesigned according to the mutation in the probe binding site. Fifty undetermined samples (2020-2021) from the previous study were retested with the newly designed probes and found to be positive for H275 and/or Y275. The results obtained were similar to the Sanger sequencing results from the previous study, suggesting that the redesigned probes were efficient in discriminating between wild-type and mutant-type viruses. Furthermore, 133 samples from 2022, making a total of 183 samples (2020-2022), were tested using improved allelic discrimination real-time RT-PCR, and the overall prevalence rate of oseltamivir resistance in 2020-2022 was found to be 0.54%.

Detection of H275Y oseltamivir resistance gene mutation among Influenza A(H1N1)pdm09 patients by allelic discrimination real-time RT-PCR.

Influenza viruses can mutate genetically and cause a range of respiratory ailments. The H275Y mutation in the neuraminidase (NA) gene reduces the effectiveness of oseltamivir, a widely used drug for the treatment of Influenza A and B virus infection. The World Health Organization (WHO) recommends single-nucleotide polymorphism assays to detect this mutation. The present study aims to estimate the prevalence of H275Y mutation conferring oseltamivir resistance in Influenza A(H1N1)pdm09 virus among hospitalized patients from June 2014 to December 2021. Following the WHO protocol, allelic discrimination real-time RT-PCR was performed for 752 samples. Out of the 752 samples, 1 tested positive for Y275 gene mutation by allelic discrimination real-time RT-PCR. In samples of years 2020 and 2021, neither the H275 nor Y275 genotype was detected. Sequencing of the NA gene of all negative samples showed a mismatch between the NA sequence and the probes used in the allelic discrimination assay. Also, Y275 mutation was detected in only 1 sample from 2020. The prevalence of oseltamivir resistance was estimated as 0.27% among the Influenza A(H1N1)pdm09 patients during 2014-2021. The study highlights that the WHO-recommended probes for detecting H275Y mutation may not be useful to detect 2020 and 2021 circulating strains of Influenza A(H1N1)pdm09, emphasizing the need for continuous monitoring of mutations in the influenza virus.

Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants.

The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

Frequency and distribution of H1N1 influenza A viruses with oseltamivir-resistant mutations worldwide before and after the 2009 pandemic.

H1N1 influenza has brought serious threats to people's health and a high socioeconomic burden to society. Oseltamivir, a kind of neuraminidase (NA) inhibitor, is the second-generation specific drug that is broadly used currently. However, H1N1 influenza viruses have exhibited oseltamivir resistance in the past decades, which might be a hidden danger. To understand the frequency and distribution laws of oseltamivir-resistant viruses, we conducted a thorough and deep analysis of the available NA protein sequences of H1N1 influenza viruses worldwide from 1918 to 2020. The differences and similarities before and after 2009 were also considered since the dominant viruses changed in this period. Results showed that 3.76% of H1N1 viruses harbored oseltamivir resistance currently. Among various significative mutations, H274Y had the highest frequency of 3.30%, while the frequencies of the other mutations were far below this whether before or after 2009. The oseltamivir resistance was mainly found in three hosts, humans, swine, and avian. Different mutation sites could exhibit different distributions in each host. Our results showed that the resistance level reached a peak during the 2007-2008 influenza season and then quickly decreased in 2009. The resistance also displayed a global distribution. The densely populated countries usually had a high resistance level. However, frequent significative mutations were also found in some small countries. Our findings indicated the necessity of monitoring oseltamivir resistance around the world. The study could provide a unique perspective toward the cognition of viruses and facilitate the future study of both pandemic and drug development.

Source of oseltamivir resistance due to single E119D and double E119D/H274Y mutations in pdm09H1N1 influenza neuraminidase.

Influenza epidemics are responsible for an average of 3-5 millions of severe cases and up to 500,000 deaths around the world. One of flu pandemic types is influenza A(H1N1)pdm09 virus (pdm09H1N1). Oseltamivir is the antiviral drug used to treat influenza targeting at neuraminidase (NA) located on the viral surface. Influenza virus undergoes high mutation rates and leads to drug resistance, and thus the development of more efficient drugs is required. In the present study, all-atom molecular dynamics simulations were applied to understand the oseltamivir resistance caused by the single E119D and double E119D/H274Y mutations on NA. The obtained results in terms of binding free energy and intermolecular interactions in the ligand-protein interface showed that the oseltamivir could not be well accommodated in the binding pocket of both NA mutants and the 150-loop moves out from oseltamivir as an "open" state. A greater number of water molecules accessible to the binding pocket could disrupt the oseltamivir binding with NA target as seen be high mobility of oseltamivir at the active site. Additionally, our finding could guide to the design and development of novel NA inhibitor drugs.

Molecular characterization of the influenza A(H1N1)pdm09 isolates collected in the 2015-2016 season and comparison of HA mutations detected in Turkey since 2009.

Influenza A(H1N1)pdm09 pandemic virus causing the 2009 global outbreak moved into the post-pandemic period, but its variants continued to be the prevailing subtype in the 2015-2016 influenza season in Europe and Asia. To determine the molecular characteristics of influenza A(H1N1)pdm09 isolates circulating during the 2015-2016 season in Turkey, we identified mutations in the hemagglutinin (HA) genes and investigated the presence of H275Y alteration in the neuraminidase genes in the randomly selected isolates. The comparison of the HA nucleotide sequences revealed a very high homology (>99.5%) among the studied influenza A(H1N1)pdm09 isolates, while a relatively low homology (96.6%-97.2%), was observed between Turkish isolates and the A/California/07/2009 vaccine virus. Overall 14 common mutations were detected in HA sequences of all 2015-2016 influenza A(H1N1)pdm09 isolates with respect to the A/California/07/2009 virus, four of which located in three different antigenic sites. Eleven rare mutations in 12 HA sequences were also detected. Phylogenetic analysis revealed that all characterized influenza A(H1N1)pdm09 isolates formed a single genetic cluster, belonging to the genetic subclade 6B.1, defined by HA amino acid substitutions S84N, S162N, and I216T. Furthermore, all isolates showed an oseltamivir-sensitive genotype, suggesting that Tamiflu (Oseltamivir) could still be the drug of choice in Turkey.

Characterization of viral genomic mutations in novel influenza A (H7N9)-infected patients: the association between oseltamivir-resistant variants and viral shedding duration.

Since February 2013, human infections with the novel influenza A H7N9 virus have occurred in eastern China. It is important to detect mutations in viral genes and analyze the clinical features of patients and viral shedding duration related to neuraminidase inhibitor (NAI) resistance. We collected clinical specimens from 31 hospitalized H7N9 patients and sequenced NA, PB2, HA, and M gene fragments. Of the 31 identified patients, 7 (22.6%) carried the R292K substitution in NA, 30 (96.8%), 3 (9.7%), and 5 (16.1%) carried E627K, Q591K, and D701N mutations in PB2, respectively, and 2 (6.5%) carried both E627K and D701N mutations in PB2. All 26 identified patients harbored Q226L mutations and possessed only a single arginine (R) at cleavage sites in the HA and a S31N mutation in M2. Among 7 NA-R292K mutated patients, 3 died and 4 were discharged. There was no significant difference in the days that patients started oseltamivir treatment after symptom onset between NA-R292K mutant and NA-R292 wild-type patients (median days, 7 vs 6, P = 0.374). NA-R292K mutant patients had a significantly longer duration of viral shedding than NA-R292 wild-type patients after oseltamivir treatment (median days, 10 vs 5, P = 0.022). The mutation of R292K in NA conferring the potential ability of oseltamivir resistance resulted in prolonged viral duration and poor outcome and should be taken into consideration in the clinical management of infected patients.

Awareness and acceptance of H1N1 vaccination among physicians: Experience of 2017 vaccination campaign.

CONTEXT: India experienced pandemic phase of H1N1 in May 2009 to December 2010. The postpandemic phase went on from January 2011 to December 2014. As per the WHO, all countries should immunize their health-care workers as a first priority to protect the essential health infrastructure. AIMS: The aim of the study is to assess the level of awareness and acceptance of influenza vaccine among physicians and also the perception of physicians regarding H1N1 infection. This study also examined time of vaccine administration in relation with efficacy concerns based on literature. SETTINGS AND DESIGN: A vaccination campaign was conducted for all health-care workers of Seth GSMC and KEM Hospital, Mumbai, in the month of July 2017 based on which a cross-sectional observational study was conducted among the physicians of the same institute. METHODS: After ethical clearance, a prevalidated pretested survey based on a pilot survey of 20 physicians was distributed among physicians, which was based on the awareness and acceptance of H1N1 vaccination among physicians and perception of H1N1 infection. Effective sample size was 272. STATISTICAL ANALYSIS USED: Descriptive statistics and Chi-square test were generated for the survey responses. All the continuous variables were reported as mean, median, and range. Categorical variables were reported as tables and pie charts. P < 0.05 was taken as significant. Data analysis was done with SPSS version 21. RESULTS: The overall vaccine compliance was 29.8%. This study has found that area of work, deficiency in knowledge about adverse effect of vaccine, misconceptions regarding vaccine, and concerns about efficacy and duration of vaccine are the important factors which lead to decreased vaccine compliance. Furthermore, it is found during the study that timing of vaccination was not given due importance as considering the epidemiological pattern. CONCLUSIONS: More emphasis should be given to education sessions and counseling of physicians regarding H1N1 vaccination and oseltamivir therapy. At administrative level, more focus should be given on timing of vaccination and other logistics. Vaccine campaigns should be conducted ideally 1 month before expected rise in cases. Quadrivalent vaccine would be more appropriate over trivalent based on epidemiology of infection in India.

Antiviral treatment and virological monitoring of oseltamivir-resistant influenza virus A(H1N1)pdm09 in a patient with chronic B lymphocytic leukemia.

We report the virological monitoring and the antiviral therapy adopted for the treatment of a patient affected by chronic B lymphocytic leukemia, who experienced a severe pneumonia with long-term shedding of influenza virus A(H1N1)pdm09, characterized by an early development of oseltamivir resistance.

Clinical management and viral genomic diversity analysis of a child's influenza A(H1N1)pdm09 infection in the context of a severe combined immunodeficiency.

INTRODUCTION: A child with severe combined immunodeficiency (SCID) had an influenza A(H1N1)pdm09 infection with viral excretion longer than 6 months, during 2013-2014 influenza season, despite cord blood transplantation and antiviral treatments. METHODS: Conventional real-time RT-PCR methods were used to estimate viral load and to detect the presence of the common N1 neuraminidase (NA) H275Y substitution responsible for oseltamivir resistance. Next-generation sequencing (NGS) of influenza viruses was performed retrospectively to characterize viral quasispecies in specimens. RESULTS: The patient was first treated with oral oseltamivir, leading to detection of low-levels of NA-H275Y substitution. Concomitant cord blood cell transplantation, intravenous administration of zanamivir and immunoglobulins led to an increase in white blood cells and influenza viral load decrease. A viral rebound occurred as soon as the antiviral treatment was discontinued. Eventually, influenza viral load was negated with immune reconstitution. NGS found influenza quasispecies harboring NA-E119A substitution (10.3%). Moreover, NGS showed that viral genomic diversity evolved under antiviral treatment and immune status. CONCLUSIONS: Conventional virological techniques were sufficient for influenza infection follow-up but NGS performances allowed characterization of viral variants evolution in this specific case of prolonged influenza virus infection. New and efficient treatments against influenza in immunocompromised patients are needed.

Emergence of Oseltamivir-Resistant H7N9 Influenza Viruses in Immunosuppressed Cynomolgus Macaques.

Antiviral compounds (eg, the neuraminidase inhibitor oseltamivir) are invaluable for the treatment of individuals infected with influenza A viruses of the H7N9 subtype (A[H7N9]), which have infected and killed hundreds of persons. However, oseltamivir treatment often leads to the emergence of resistant viruses in immunocompromised individuals. To better understand the emergence and properties of oseltamivir-resistant A(H7N9) viruses in immunosuppressed individuals, we infected immunosuppressed cynomolgus macaques with an A(H7N9) virus and treated them with oseltamivir. Disease severity and mortality were higher in immunosuppressed than in immunocompetent animals. Oseltamivir treatment at 2 different doses reduced A(H7N9) viral titers in infected animals, but even high-dose oseltamivir did not block viral replication sufficiently to suppress the emergence of resistant variants. Some resistant variants were not appreciably attenuated in cultured cells, but an oseltamivir-resistant A(H7N9) virus did not transmit among ferrets. These findings are useful for the control of A(H7N9) virus infections in clinical settings.

Characterization of oseltamivir-resistant influenza virus populations in immunosuppressed patients using digital-droplet PCR: Comparison with qPCR and next generation sequencing analysis.

INTRODUCTION: The H275Y substitution in neuraminidase (NA) confers oseltamivir-resistance in A(H1N1) influenza viruses (IV). Droplet digital PCR (ddPCR) is a new technique to explore single nucleotide polymorphisms. The aim of this study was to compare the performances of reverse transcriptase (RT)-ddPCR, RT-qPCR and next generation sequencing (NGS). We also analyzed the proportions of H275Y-NA substitution for two immunosuppressed patients with sustained shedding of A(H1N1)pdm09 IV. METHODS: RT-qPCR was performed using the ABI7500 platform. RT-ddPCR was carried out using the QX200 ddPCR platform. We strengthened our results by a NGS assay (Ion PGM™ sequencer). Discrimination performance and sensitivity of the RT-ddPCR assay were evaluated using mixes of wild type (WT) and mutated H275Y-NA-coding segments. RESULTS: The performance of RT-ddPCR was better than RT-qPCR, using NGS assay as a gold standard. RT-ddPCR was able to detect 0.28% oseltamivir-resistant IV in a WT IV population and 0.55% WT IV in an oseltamivir-resistant IV population. For the first patient, the H275Y-NA substitution was selected by oseltamivir treatment and reached about 50% of the IV population before dropping to less than 2% after treatment discontinuation which was under the lower limit of quantification by RT-qPCR and RT-ddPCR (<2%) after treatment stop. Then, five days after oseltamivir was re-introduced, the H275Y-NA substitution rose up to 100%. For the second patient, the H275Y-NA substitution reached about 30% two days after oseltamivir discontinuation. CONCLUSION: RT-ddPCR demonstrated better performances than classical RT-qPCR to estimate oseltamivir-resistant IV proportions. This technique could be used to detect earlier emergence of H275Y-NA substitution.

Human Infection with Highly Pathogenic Avian Influenza A(H7N9) Virus, China.

The recent increase in zoonotic avian influenza A(H7N9) disease in China is a cause of public health concern. Most of the A(H7N9) viruses previously reported have been of low pathogenicity. We report the fatal case of a patient in China who was infected with an A(H7N9) virus having a polybasic amino acid sequence at its hemagglutinin cleavage site (PEVPKRKRTAR/GL), a sequence suggestive of high pathogenicity in birds. Its neuraminidase also had R292K, an amino acid change known to be associated with neuraminidase inhibitor resistance. Both of these molecular features might have contributed to the patient's adverse clinical outcome. The patient had a history of exposure to sick and dying poultry, and his close contacts had no evidence of A(H7N9) disease, suggesting human-to-human transmission did not occur. Enhanced surveillance is needed to determine whether this highly pathogenic avian influenza A(H7N9) virus will continue to spread.

A 3D-RISM/RISM study of the oseltamivir binding efficiency with the wild-type and resistance-associated mutant forms of the viral influenza B neuraminidase.

The binding affinity of oseltamivir to the influenza B neuraminidase and to its variants with three single substitutions, E119G, R152K, and D198N, is investigated by the MM/3D-RISM method. The binding affinity or the binding free energy of ligand to receptor was found to be determined by a subtle balance of two major contributions that largely cancel out each other: the ligand-receptor interactions and the dehydration free energy. The theoretical results of the binding affinity of the drug to the mutants reproduced the observed trend in the resistivity, measured by IC50 ; the high-level resistance of E119G and R152K, and the low-level resistance of D198N. For E119G and R152K, reduction of the direct drug-target interaction, especially at the mutated residue, is the main source of high-level oseltamivir resistance. This phenomenon, however, is not found in the D198N strain, which is located in the framework of the active-site.

Circulación de virus de influenza A y cepas resistentes a oseltamivir en el período invernal del año 2013 / Circulation of Influenza A Viruses and Oseltamivir-Resistant Strains during the 2013 Winter

Inicialmente el virus de influenza A(H1N1)pdm09 fue susceptible a los inhibidores de neuraminidasa oseltamivir y zanamivir. Las cepas virales resistentes presentan una sustitución que produce un cambio del aminoácido histidina (H) por una tirosina (Y) en el codón 274 del gen de la neuroaminidasa. El objetivo del trabajo fue realizar un análisis retrospectivo de los resultados obtenidos en muestras estudiadas para influenza A durante el periodo junio - agosto de 2013 y en las muestras positivas determinar la presencia de la mutación H274Y. Se estudiaron 1783 muestras de pacientes con diagnóstico presuntivo de influenza A(H1N1)pdm09, 171 (9.6%) resultaron positivas, a estas se les estudió la presencia de la mutación H274Y. Únicamente dos muestras presentaron la mutación de resistencia. Los métodos para detectar cepas de infuenza A(H1N1)pdm09 resistentes son necesarios para ayudar a los médicos en la selección de la terapia antiviral apropiada de la influenza.

Initially the circulating influenza virus A(H1N1)pdm09 was susceptible to neuraminidase inhibitors oseltamivir and zanamivir. Virtually all resistant viruses possess a substitution at codon 274 of the neuraminidase gene which produces a change of the amino acid histidine (H) to a tyrosine (Y). The aims of the study were to perform a retrospective analysis of samples studied in the Laboratory of Genomic Medicine - MANLAB for influenza A during the period June to August 2013 in Buenos Aires, and to determine the presence of the H274Y mutation. 1783 samples from patients with a presumptive diagnosis of influenza A(H1N1) pdm09 were studied, the virus was detected in 171 samples (9.6%). Then, we studied the presence of the mutation H274Y. Only two samples showed the characteristic resistance mutation. Methods for detecting oseltamivir-resistant A(H1N1)pdm09 influenza strains are needed to assist physicians in the selection of appropriate antiviral therapy for influenza treatment.

Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir resistant H1N1 influenza viruses: modeling studies of antibody and receptor binding.

The envelope protein hemagglutinin (HA) of influenza viruses is primarily associated with host antibody and receptor interactions. The HA protein is known to maintain a functional balance with neuraminidase (NA), the other major envelope protein. Prior to 2007-2008, human seasonal H1N1 viruses possessing the NA H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. Subsequently, secondary mutations that compensate for the deleterious effect of the NA H274Y mutation have been identified. The molecular mechanism of how the defect could be counteracted by these secondary mutations is not fully understood. We studied here the effect of three such mutations (T86K, K144E and R192K) in the HA protein, which are located at either the HA receptor binding site or in the H1N1 antigenic sites. Molecular docking and dynamics studies showed that, of the three mutations, the R192K mutation could have mediated neutralizing antibody escape and decreased receptor binding affinity, either or both of which may have contributed to increased viral fitness. The study suggests the molecular basis of enhanced viral fitness induced by secondary mutations in the evolution of oseltamivir-resistant influenza strains.

Molecular findings from influenza A(H1N1)pdm09 detected in patients from a Brazilian equatorial region during the pandemic period

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.

Development and clinical testing of a simple, low-density gel element array for influenza identification, subtyping, and H275Y detection.

The objectives of this study were to develop a user-friendly, gel element microarray test for influenza virus detection, subtyping, and neuraminidase inhibitor resistance detection, assess the performance characteristics of the assay, and perform a clinical evaluation on retrospective nasopharyngeal swab specimens. A streamlined microarray workflow enabled a single user to run up to 24 tests in an 8h shift. The most sensitive components of the test were the primers and probes targeting the A/H1 pdm09 HA gene with an analytical limit of detection (LoD) <100 gene copies (gc) per reaction. LoDs for all targets in nasopharyngeal swab samples were ≤1000 gc, with the exception of one target in the seasonal A/H1N1 subtype. Seasonal H275Y variants were detectable in a mixed population when present at >5% with wild type virus, while the 2009 pandemic H1N1 H275Y variant was detectable at ≤1% in a mixture with pandemic wild type virus. Influenza typing and subtyping results concurred with those obtained with real-time RT-PCR assays on more than 97% of the samples tested. The results demonstrate that a large panel of single-plex, real-time RT-PCR tests can be translated to an easy-to-use, sensitive, and specific microarray test for potential diagnostic use.

A novel I221L substitution in neuraminidase confers high-level resistance to oseltamivir in influenza B viruses.

UNLABELLED: Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment. METHODS: Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type or mutated (I221L) NA genes were evaluated. Proportions of wild-type and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wild-type and I221L variant NA were performed. RESULTS: The Km and Ki revealed that the I221L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir, respectively, compared with wild-type NA. Viruses with a wild-type or I221L variant NA had similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, and 5 passages in MDCK cells revealed no reversion of the I221L substitution. The crystal structure of the I221L NA and oseltamivir complex showed that the leucine side chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir. CONCLUSIONS: Enzyme kinetic and NA structural analyses provide an explanation for the high level of resistance to oseltamivir while retaining good fitness of viruses carrying I221L variant NA.