VPipe: an Automated Bioinformatics Platform for Assembly and Management of Viral Next-Generation Sequencing Data.

Next-generation sequencing (NGS) is a powerful tool for detecting and investigating viral pathogens; however, analysis and management of the enormous amounts of data generated from these technologies remains a challenge. Here, we present VPipe (the Viral NGS Analysis Pipeline and Data Management System), an automated bioinformatics pipeline optimized for whole-genome assembly of viral sequences and identification of diverse species. VPipe automates the data quality control, assembly, and contig identification steps typically performed when analyzing NGS data. Users access the pipeline through a secure web-based portal, which provides an easy-to-use interface with advanced search capabilities for reviewing results. In addition, VPipe provides a centralized system for storing and analyzing NGS data, eliminating common bottlenecks in bioinformatics analyses for public health laboratories with limited on-site computational infrastructure. The performance of VPipe was validated through the analysis of publicly available NGS data sets for viral pathogens, generating high-quality assemblies for 12 data sets. VPipe also generated assemblies with greater contiguity than similar pipelines for 41 human respiratory syncytial virus isolates and 23 SARS-CoV-2 specimens. IMPORTANCE Computational infrastructure and bioinformatics analysis are bottlenecks in the application of NGS to viral pathogens. As of September 2021, VPipe has been used by the U.S. Centers for Disease Control and Prevention (CDC) and 12 state public health laboratories to characterize >17,500 and 1,500 clinical specimens and isolates, respectively. VPipe automates genome assembly for a wide range of viruses, including high-consequence pathogens such as SARS-CoV-2. Such automated functionality expedites public health responses to viral outbreaks and pathogen surveillance.

Web-Based Genome Analysis of Bacterial Meningitis Pathogens for Public Health Applications Using the Bacterial Meningitis Genomic Analysis Platform (BMGAP).

Effective laboratory-based surveillance and public health response to bacterial meningitis depends on timely characterization of bacterial meningitis pathogens. Traditionally, characterizing bacterial meningitis pathogens such as Neisseria meningitidis (Nm) and Haemophilus influenzae (Hi) required several biochemical and molecular tests. Whole genome sequencing (WGS) has enabled the development of pipelines capable of characterizing the given pathogen with equivalent results to many of the traditional tests. Here, we present the Bacterial Meningitis Genomic Analysis Platform (BMGAP): a secure, web-accessible informatics platform that facilitates automated analysis of WGS data in public health laboratories. BMGAP is a pipeline comprised of several components, including both widely used, open-source third-party software and customized analysis modules for the specific target pathogens. BMGAP performs de novo draft genome assembly and identifies the bacterial species by whole-genome comparisons against a curated reference collection of 17 focal species including Nm, Hi, and other closely related species. Genomes identified as Nm or Hi undergo multi-locus sequence typing (MLST) and capsule characterization. Further typing information is captured from Nm genomes, such as peptides for the vaccine antigens FHbp, NadA, and NhbA. Assembled genomes are retained in the BMGAP database, serving as a repository for genomic comparisons. BMGAP's species identification and capsule characterization modules were validated using PCR and slide agglutination from 446 bacterial invasive isolates (273 Nm from nine different serogroups, 150 Hi from seven different serotypes, and 23 from nine other species) collected from 2017 to 2019 through surveillance programs. Among the validation isolates, BMGAP correctly identified the species for all 440 isolates (100% sensitivity and specificity) and accurately characterized all Nm serogroups (99% sensitivity and 98% specificity) and Hi serotypes (100% sensitivity and specificity). BMGAP provides an automated, multi-species analysis pipeline that can be extended to include additional analysis modules as needed. This provides easy-to-interpret and validated Nm and Hi genome analysis capacity to public health laboratories and collaborators. As the BMGAP database accumulates more genomic data, it grows as a valuable resource for rapid comparative genomic analyses during outbreak investigations.

Emergence of Multidrug-Resistant Influenza A(H1N1)pdm09 Virus Variants in an Immunocompromised Child Treated With Oseltamivir and Zanamivir.

Prolonged treatment of an immunocompromised child with oseltamivir and zanamivir for A(H1N1)pdm09 virus infection led to the emergence of viruses carrying H275Y and/or E119G in the neuraminidase (NA). When phenotypically evaluated by NA inhibition, the dual H275Y-E119G substitution caused highly reduced inhibition by 4 NA inhibitors: oseltamivir, zanamivir, peramivir, and laninamivir.

Application of a seven-target pyrosequencing assay to improve the detection of neuraminidase inhibitor-resistant Influenza A(H3N2) viruses.

National U.S. influenza antiviral surveillance incorporates data generated by neuraminidase (NA) inhibition (NI) testing of isolates supplemented with NA sequence analysis and pyrosequencing analysis of clinical specimens. A lack of established correlates for clinically relevant resistance to NA inhibitors (NAIs) hinders interpretation of NI assay data. Nonetheless, A(H3N2) viruses are commonly monitored for moderately or highly reduced inhibition in the NI assay and/or for the presence of NA markers E119V, R292K, and N294S. In 2012 to 2013, three drug-resistant A(H3N2) viruses were detected by NI assay among isolates (n = 1,424); all showed highly reduced inhibition by oseltamivir and had E119V. In addition, one R292K variant was detected among clinical samples (n = 1,024) by a 3-target pyrosequencing assay. Overall, the frequency of NAI resistance was low (0.16% [4 of 2,448]). To screen for additional NA markers previously identified in viruses from NAI-treated patients, the pyrosequencing assay was modified to include Q136K, I222V, and deletions encompassing residues 245 to 248 (del245-248) and residues 247 to 250 (del247-250). The 7-target pyrosequencing assay detected NA variants carrying E119V, Q136, and del245-248 in an isolate from an oseltamivir-treated patient. Next, this assay was applied to clinical specimens collected from hospitalized patients and submitted for NI testing but failed cell culture propagation. Of the 27 clinical specimens tested, 4 (15%) contained NA changes: R292K (n = 2), E119V (n = 1), and del247-250 (n = 1). Recombinant NAs with del247-250 or del245-248 conferred highly reduced inhibition by oseltamivir, reduced inhibition by zanamivir, and normal inhibition by peramivir and laninamivir. Our results demonstrated the benefits of the 7-target pyrosequencing assay in conducting A(H3N2) antiviral surveillance and testing for clinical care.

Oseltamivir-resistant influenza A(H1N1)pdm09 viruses, United States, 2013-14.

We report characteristics of oseltamivir-resistant influenza A(H1N1)pdm09 viruses and patients infected with these viruses in the United States. During 2013-14, fifty-nine (1.2%) of 4,968 analyzed US influenza A(H1N1)pdm09 viruses had the H275Y oseltamivir resistance-conferring neuraminidase substitution. Our results emphasize the need for local surveillance for neuraminidase inhibitor susceptibility among circulating influenza viruses.

Drug susceptibility surveillance of influenza viruses circulating in the United States in 2011-2012: application of the WHO antiviral working group criteria.

BACKGROUND: Assessing susceptibility of influenza viruses to neuraminidase (NA) inhibitors (NAIs) is primarily done in NA inhibition (NI) assays, supplemented by NA sequence analysis. However, two factors present challenges for NI assay data interpretation: lack of established IC50 values indicative of clinically relevant resistance and insufficient harmonization of NI testing methodologies among surveillance laboratories. In 2012, the WHO working group on influenza antiviral susceptibility (WHO-AVWG) developed criteria to facilitate consistent interpretation and reporting of NI assay data. METHODS: The WHO-AVWG classification criteria were applied in interpreting NI assay data for two FDA-licensed NAIs, oseltamivir and zanamivir, for viruses collected in the United States during the 2011-2012 winter season. RESULTS: All A (H1N1)pdm09 viruses (n = 449) exhibited normal inhibition by oseltamivir and zanamivir, with the exception of eight viruses (1·8%) with highly reduced inhibition by oseltamivir, which carried the H275Y marker of oseltamivir resistance. A (H3N2) viruses (n = 978) exhibited normal inhibition by both NAIs, except for one virus with highly reduced inhibition by zanamivir due to the cell culture-selected NA change, Q136K. Type B viruses (n = 343) exhibited normal inhibition by both drugs, except for an isolate with reduced inhibition by both NAIs that had the cell culture-selected A200T substitution. CONCLUSIONS: WHO-AVWG classification criteria allowed the detection of viruses carrying the established oseltamivir resistance marker, as well as viruses whose susceptibility was altered during propagation. These criteria were consistent with statistical-based criteria for detecting outliers and will be useful in harmonizing NI assay data among surveillance laboratories worldwide and in establishing laboratory correlates of clinically relevant resistance.

Cell culture-selected substitutions in influenza A(H3N2) neuraminidase affect drug susceptibility assessment.

Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC50) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n = 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.

Bioluminescence-based neuraminidase inhibition assay for monitoring influenza virus drug susceptibility in clinical specimens.

The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n = 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50% inhibitory concentrations (IC50s) generated were compared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof of principle, clinical specimens (n = 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyrosequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens.

Neuraminidase inhibitor susceptibility surveillance of influenza viruses circulating worldwide during the 2011 Southern Hemisphere season.

BACKGROUND: Neuraminidase (NA) inhibitors (NAIs) are currently the only antivirals effective against influenza infections due to widespread resistance to M2 inhibitors. METHODS: Influenza A and B viruses (n = 1079) collected worldwide between April 01, 2011, and September 30, 2011, were assessed for susceptibility to FDA-approved NAIs, oseltamivir and zanamivir, and investigational peramivir, using the fluorescent-based NA-Fluor™ Influenza Neuraminidase Assay Kit. A subset of viruses (n = 98) were tested for susceptibility to the investigational NAI, laninamivir. RESULTS: Influenza A(H1N1)pdm09 viruses (n = 326) were sensitive to all NAIs, except for two (0.6%) with H275Y (N1 numbering; H274Y in N2 numbering) substitution, which exhibited elevated IC50 s for oseltamivir and peramivir, and a third with previously unreported N325K substitution, exhibiting reduced susceptibility to oseltamivir. Influenza A(H3N2) viruses (n = 407) were sensitive to all NAIs. Influenza B viruses (n = 346) were sensitive to all NAIs, except two (0.6%) with H273Y (N1 numbering; H274Y in N2 numbering) substitution, exhibiting reduced susceptibility to oseltamivir and peramivir, and one with previously unreported G140R and N144K substitutions, exhibiting reduced susceptibility to oseltamivir, zanamivir, and peramivir. All influenza A and B viruses were sensitive to laninamivir. It is unknown whether substitutions N325K, G140R, and N144K were present in the virus prior to culturing because clinical specimens were unavailable for testing. CONCLUSIONS: This study summarizes NAI susceptibility of influenza viruses circulating worldwide during the 2011 Southern Hemisphere (SH) season, assessed using the NA-Fluor™ Kit. Despite low resistance to NAIs among tested influenza viruses, constant surveillance of influenza virus susceptibility to NAIs should be emphasized.

Assays for monitoring susceptibility of influenza viruses to neuraminidase inhibitors.

Close monitoring of drug susceptibility among human influenza viruses was necessitated by widespread resistance to M2 inhibitors in influenza H1N1 (pre-pandemic and 2009 pandemic) and H3N2 viruses, and of oseltamivir resistance in pre-pandemic H1N1 viruses. The FDA-approved neuraminidase (NA) inhibitors (NAIs), oseltamivir and zanamivir, as well as investigational NAIs, peramivir and laninamivir, are currently the principal treatment options for managing influenza infection. However, there are challenges associated with assessing virus susceptibility to this class of drugs. Traditional cell culture-based assays are not reliable for phenotypic testing of NAI susceptibility due to complexity in interpretation. Two types of laboratory assays are currently available for monitoring NAI susceptibility, phenotypic such as the neuraminidase inhibition (NI) assay and genotypic. The NI assay's requirement for propagated virus lengthens testing turnaround; therefore, the need for timely detection of molecular markers associated with NAI resistance (e.g., H275Y in H1N1) has spurred the development of rapid, high-throughput assays, such as real-time RT-PCR and pyrosequencing. The high sensitivity of genotypic assays allows testing of clinical specimens thus eliminating the need for virus propagation in cell culture. The NI assays are especially valuable when a novel virus emerges or a new NAI becomes available. Modifications continue to be introduced into NI assays, including optimization and data analysis criteria. The optimal assay of choice for monitoring influenza drug susceptibility varies widely depending on the needs of laboratories (e.g., surveillance purposes, clinical settings). Optimally, it is desirable to combine functional and genetic analyses of virus isolates and, when possible, the respective clinical specimens.

The chemiluminescent neuraminidase inhibition assay: a functional method for detection of influenza virus resistance to the neuraminidase inhibitors.

Neuraminidase inhibitors (NAIs) represent a newer class of anti-influenza drugs. Widespread natural or acquired resistance to NAIs is a major public health concern as it limits pharmaceutical options available for managing seasonal and pandemic influenza virus infections. Molecular-based methods, such as pyrosequencing, sequencing, and PCR are rapid techniques for detecting known genetic markers of resistance, but they are unable to identify novel mutations that may confer resistance, or subtle differences in the susceptibility of viruses to the NAIs. This chapter describes the chemiluminescent neuraminidase (NA) inhibition (NI) assay, a functional method used for assessing influenza virus susceptibility to NAIs. The assay generates IC(50) values (drug concentration needed to reduce the NA enzymatic activity by 50%) which are determined by curve-fitting analysis. Test viruses showing elevated IC(50) values relative to those of NAI-sensitive reference viruses of the same antigenic type and subtype are further analyzed by pyrosequencing or conventional sequencing to identify known markers of NAI resistance or new changes in the NA. The criteria for NAI resistance are currently not well defined and tend to vary by laboratory and NI assay, therefore harmonization of NI assay conditions and interpretation of results across surveillance laboratories is necessary to improve the NAI susceptibility testing and analysis.

The fluorescence neuraminidase inhibition assay: a functional method for detection of influenza virus resistance to the neuraminidase inhibitors.

Neuraminidase inhibitors (NAIs) are presently the only effective antiviral drugs for treatment and chemoprophylaxis of influenza A and B infections, due to the high prevalence of resistance to the adamantane class of drugs among influenza A(H3N2) and A(H1N1) viruses, including the 2009 pandemic H1N1 strain. The limited pharmaceutical options currently available for control of influenza infections underscore the critical need for surveillance on NAI susceptibility of influenza viruses circulating globally. This chapter describes the fluorescent neuraminidase (NA) inhibition (NI) assay, a functional method used for assessing influenza virus susceptibility to NAIs. The IC(50) (drug concentration needed to reduce the NA enzymatic activity by 50%) values generated in this assay are used to evaluate the NAI-susceptibility of test viruses relative to those of sensitive reference viruses of the same antigenic type and subtype. Test viruses with significantly elevated IC(50)s are further analyzed by pyrosequencing or conventional sequencing to identify known markers of NAI resistance or novel changes in the NA. The harmonization of NI assay conditions and interpretation of results across surveillance laboratories is necessary to improve NAI susceptibility testing and analysis.

Oseltamivir-resistant pandemic (H1N1) 2009 virus infections, United States, 2010-11.

During October 2010-July 2011, 1.0% of pandemic (H1N1) 2009 viruses in the United States were oseltamivir resistant, compared with 0.5% during the 2009-10 influenza season. Of resistant viruses from 2010-11 and 2009-10, 26% and 89%, respectively, were from persons exposed to oseltamivir before specimen collection. Findings suggest limited community transmission of oseltamivir-resistant virus.

Characteristics of patients with oseltamivir-resistant pandemic (H1N1) 2009, United States.

During April 2009-June 2010, thirty-seven (0.5%) of 6,740 pandemic (H1N1) 2009 viruses submitted to a US surveillance system were oseltamivir resistant. Most patients with oseltamivir-resistant infections were severely immunocompromised (76%) and had received oseltamivir before specimen collection (89%). No evidence was found for community circulation of resistant viruses; only 4 (unlinked) patients had no oseltamivir exposure.

Comprehensive assessment of 2009 pandemic influenza A (H1N1) virus drug susceptibility in vitro.

BACKGROUND: Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010. METHODS: Virus isolates were tested for adamantane susceptibility, using pyrosequencing to detect the S31N marker of adamantane resistance in the M2 protein and biological assays to assess viral replication in cell culture. To assess neuraminidase (NA) inhibitor (NAI) susceptibility, virus isolates were tested in chemiluminescent NA inhibition assays and by pyrosequencing to detect the H275Y (H274Y in N2 numbering) marker of oseltamivir resistance in the NA. RESULTS: With the exception of three, all viruses that were tested for adamantane susceptibility (n=3,362) were resistant to this class of drugs. All viruses tested for NAI susceptibility (n=3,359) were sensitive to two US Food and Drug Administration-approved NAIs, oseltamivir (mean ±sd 50% inhibitory concentration [IC(50)] 0.25 ±0.12 nM) and zanamivir (mean IC(50) 0.29 ±0.09 nM), except 23 (0.7%), which were resistant to oseltamivir, but sensitive to zanamivir. Oseltamivir-resistant viruses had the H275Y mutation in their NA and were detected in patients exposed to the drug through prophylaxis or treatment. NA activity of all viruses was inhibited by the NAIs peramivir, laninamivir (R-125489) and A-315675, except for H275Y variants, which exhibited approximately 100-fold reduction in peramivir susceptibility. CONCLUSIONS: This report provides data regarding antiviral susceptibility of 2009 pandemic influenza A (H1N1) surveillance viruses, the majority of which were resistant to adamantanes and sensitive to NAIs. These findings provide information essential for antiviral resistance monitoring and development of novel diagnostic tests for detecting influenza antiviral resistance.

Detection of E119V and E119I mutations in influenza A (H3N2) viruses isolated from an immunocompromised patient: challenges in diagnosis of oseltamivir resistance.

The clinical use of the neuraminidase inhibitor (NAI) oseltamivir is associated with the emergence of drug resistance resulting from subtype-specific neuraminidase (NA) mutations. The influenza A/Texas/12/2007 (H3N2) virus isolated from an oseltamivir-treated immunocompromised patient exhibited reduced susceptibility to oseltamivir in the chemiluminescent neuraminidase inhibition (NI) assay (approximately 60-fold increase in its 50% inhibitory concentration [IC(50)] compared to that for a control virus). When further propagated in cell culture, the isolate maintained reduced susceptibility to oseltamivir in both chemiluminescent and fluorescent NI assays (approximately 50- and 350-fold increases in IC(50), respectively). Sequencing analysis of the isolate revealed a mix of nucleotides coding for amino acids at position 119 of the NA [E119(V/I)]. Plaque purification of the isolate yielded E119V and E119I variants, both exhibiting reduced susceptibility to oseltamivir. The E119I variant also showed decreased susceptibility to zanamivir and the investigational NAIs peramivir and A-315675. The emergence of E119V variants in oseltamivir-treated patients has been previously reported; however, the E119I mutation detected here is a novel one which reduces susceptibility to several NAIs. Both mutations were not detected in unpropagated original clinical specimens using either conventional sequencing or pyrosequencing, suggesting that these variants were present in very low proportions (<10%) in clinical specimens and gained dominance after virus propagation in MDCK cells. All virus isolates recovered from the patient were resistant to adamantanes. Our findings highlight the potential for emergence and persistence of multidrug-resistant influenza viruses in oseltamivir-treated immunocompromised subjects and also highlight challenges for drug resistance diagnosis due to the genetic instability of the virus population upon propagation in cell culture.

Neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective.

Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n = 5540) collected during the 2008-2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC(50)s were performed for each virus type/subtype and NAI to identify outliers which were characterized based on a statistical cutoff of IC(50) >3 interquartile ranges (IQR) from the 75(th) percentile. Among 1533 seasonal H1N1 viruses tested, 1431 (93.3%) were outliers for oseltamivir; they all harbored the H275Y mutation in the neuraminidase (NA) and were reported as oseltamivir-resistant. Only 15 (0.7%) of pandemic 2009 H1N1 viruses tested (n = 2259) were resistant to oseltamivir. All influenza A(H3N2) (n = 834) and B (n = 914) viruses were sensitive to oseltamivir, except for one A(H3N2) and one B virus, with D151V and D197E (D198E in N2 numbering) mutations in the NA, respectively. All viruses tested were sensitive to zanamivir, except for six seasonal A(H1N1) and several A(H3N2) outliers (n = 22) which exhibited cell culture induced mutations at residue D151 of the NA. A subset of viruses (n = 1058) tested for peramivir were sensitive to the drug, with exception of H275Y variants that exhibited reduced susceptibility to this NAI. This study summarizes baseline susceptibility patterns of seasonal and pandemic influenza viruses, and seeks to contribute towards criteria for defining NAI resistance.

Host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in A(H1N1) viruses.

The neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, are essential for treatment and prevention of influenza A and B infections. Oseltamivir resistance among influenza A (H1N1) viruses rapidly emerged and spread globally during the 2007-2008 and 2008-2009 influenza seasons. Approximately 20% and 90% of viruses tested for NAI susceptibility at CDC during these seasons, respectively, were resistant to oseltamivir (IC(50) approximately 100-3000 time>those of sensitive viruses), based on the chemiluminescent NA inhibition assay. Pyrosequencing analysis confirmed H274Y mutation (H275Y in N1 numbering) in the neuraminidase (NA) gene of oseltamivir-resistant viruses. Full NA sequence analysis of a subset of oseltamivir-resistant and sensitive virus isolates from both seasons (n=725) showed that 53 (7.3%) had mutations at residue D151 (D-->E/G/N), while 9 (1.2%) had mutations at Q136 (Q-->K) and 2 (0.3%) had mutations at both residues. Viruses with very high IC(50) for oseltamivir and peramivir, and elevated IC(50) for zanamivir, had H274Y in addition to mutations at D151 and/or Q136, residues which can potentially confer NAI resistance based on recent N1 NA crystal structure data. Mutations at D151 without H274Y, did not elevate IC(50) for any tested NAI, however, Q136K alone significantly reduced susceptibility to zanamivir (36-fold), peramivir (80-fold) and A-315675 (114-fold) but not oseltamivir. Mutations at D151 and Q136 were present only in MDCK grown viruses but not in matching original clinical specimens (n=33) which were available for testing, suggesting that these variants were the result of cell culture selection or they were present in very low proportions. Our findings provide evidence that propagation of influenza virus outside its natural host may lead to selection of virus variants with mutations in the NA that affect sensitivity to NAIs and thus poses implications for drug resistance monitoring and diagnostics.

Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing.

The M2 blockers amantadine and rimantadine and the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir are approved by the FDA for use for the control of influenza A virus infections. The 2009 pandemic influenza A (H1N1) viruses (H1N1pdm) are reassortants that acquired M and NA gene segments from a Eurasian adamantane-resistant swine influenza virus. NAI resistance in the H1N1pdm viruses has been rare, and its occurrence is mainly limited to oseltamivir-exposed patients. The pyrosequencing assay has been proven to be a useful tool in surveillance for drug resistance in seasonal influenza A viruses. We provide a protocol which allows the detection of adamantane resistance markers as well as the I43T change, which is unique to the H1N1pdm M2 protein. The protocol also allows the detection of changes at residues V116, I117, E119, Q136, K150, D151, D199, I223, H275, and N295 in the NA, known to alter NAI drug susceptibility. We report on the detection of the first cases of the oseltamivir resistance-conferring mutation H275Y and the I223V change in viruses from the United States using the approach described in this study. Moreover, the assay permits the quick identification of the major NA group (V106/N248, I106/D248, or I106/N248) to which a pandemic virus belongs. Pyrosequencing is well suited for the detection of drug resistance markers and signature mutations in the M and NA gene segments of the pandemic H1N1 influenza viruses.

Rapid identification of oseltamivir-resistant influenza A(H1N1) viruses with H274Y mutation by RT-PCR/restriction fragment length polymorphism assay.

In the beginning of 2007-2008 Northern Hemisphere influenza season, the frequency of influenza A(H1N1) viruses bearing a previously defined oseltamivir resistance conferring amino acid change of Histidine to Tyrosine at position 274 (H274Y) of the neuraminidase (NA) increased dramatically. In order to rapidly detect such resistant viruses, an RT-PCR/restriction fragment length polymorphism (RT-PCR/RFLP) assay targeting amino acid 274 of the N1 NA molecule was developed to investigate the presence or absence of the H274Y mutation. The reverse primer was engineered to produce a BspHI site in the amplicon for oseltamivir-sensitive viruses with Histidine at position 274 (274H). A total of 50 influenza A(H1N1) specimens including 30 oseltamivir-sensitive and 20 oseltamivir-resistant ones submitted to the Centers for Disease Control and Prevention (CDC) during the 2007-2008 influenza season were successfully characterized by this assay. The assay was specific for grown A(H1N1) viruses and original clinical specimens, with a lower limit of detection of approximately 10 RNA transcript copies per reaction. Our RT-PCR/RFLP assay provides a simple, rapid and sensitive tool to monitor the emergence and spread of H274Y oseltamivir-resistant influenza A(H1N1) viruses.