Insight into the Oseltamivir Resistance R292K Mutation in H5N1 Influenza Virus: A Molecular Docking and Molecular Dynamics Approach

H5N1 is a subtype of the influenza A virus that can cause disease in humans and many other animal species. Oseltamivir (Tamiflu) is a potent and selective antiviral drug employed to fight the flu virus in infected individuals by inhibiting neuraminidase (NA), a flu protein responsible for the release and spread of the progeny virions. However, oseltamivir resistance has become a critical problem. In particular, influenza strains with a R292K NA mutation are highly resistant to the oseltamivir. Though the biological functions of the mutations have previously been characterized, the structural basis behind the reduced catalytic activity and reduced protein level is not clear. In this study, molecular docking and molecular dynamics (MD) approach were employed to investigate the structural and dynamical effects throughout the protein structure and specifically, at the drug-binding pocket. Furthermore, potential of mean force was analyzed using explicit solvent MD simulations with the umbrella sampling method to explore the free energy of binding. It is believed that this study provides valuable guidance for the resistance management of oseltamivir and designing of more potent antiviral inhibitor.     

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

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

Hyphenated 3D-QSAR statistical model-drug repurposing analysis for the identification of potent neuraminidase inhibitor

The Influenza A virus is one of the principle causes of respiratory illness in human. The surface glycoprotein of the influenza virus, neuraminidase (NA), has a vital role in the release of new viral particle and spreads infection in the respiratory tract. It has been long recognized as a valid drug target for influenza A virus infection. Oseltamivir is used as a standard drug of choice for the treatment of influenza. However, the emergence of mutants with novel mutations has increased the resistance to potent NA inhibitor. In the present investigation, we have employed computer-assisted combinatorial techniques in the screening of 8621 molecules from Drug Bank to find potent NA inhibitors. A three-dimensional pharmacophore model was generated from the previously reported 28 carbocylic influenza NA inhibitors along with oseltamivir using PHASE module of Schrödinger Suite. The model generated consists of one hydrogen bond acceptor (A), one hydrogen bond donors (D), one hydrophobic group (H), and one positively charged group (P), ADHP. The hypothesis was further validated for its integrity and significance using enrichment analysis. Subsequently, an atom-based 3D-QSAR model was built using the common pharmacophore hypothesis (CPH). The developed 3D-QSAR model was found to be statistically significant with R² value of 0.9866 and Q² value of 0.7629. Further screening was accomplished using three-stage docking process using the Glide algorithm. The resultant lead molecules were examined for its drug-like properties using the Qikprop algorithm. Finally, the calculated pIC₅₀ values of the lead compounds were validated by the AutoQSAR algorithm. Overall, the results from our analysis highlights that lisinopril (DB00722) is predicted to bind better with NA than currently approved drug. In addition, it has the best match in binding geometry conformations with the existing NA inhibitor. Note that the antiviral activity of lisinopril is reported in the literature. However, our paper is the first report on lisinopril activity against influenza A virus infection. These results are envisioned to help design the novel NA inhibitors with an increased antiviral efficacy.     

In silico analysis of drug-resistant mutant of neuraminidase (N294S) against oseltamivir

The recent H1N1 influenza pandemic has attracted worldwide attention due to the high infection rate. Oseltamivir is a new class of anti-viral agent approved for the treatment and prevention of influenza infections. The principal target for this drug is a virus surface glycoprotein, neuraminidase (NA), which facilitates the release of nascent virus and thus spreads infection. Until recently, only a low prevalence of neuraminidase inhibitor (NAI) resistance (<1 %) had been detected in circulating viruses. However, there have been reports of significant numbers of A (H1N1) influenza strains with a N294S neuraminidase mutation that was highly resistant to the NAI, oseltamivir. Hence, in the present study, we highlight the effect of point mutation-induced oseltamivir resistance in H1N1 subtype neuraminidases by molecular simulation approach. The docking analysis reveals that mutation (N294S) significantly affects the binding affinity of oseltamivir with mutant type NA. This is mainly due to the decrease in the flexibility of binding site residues and the difference in prevalence of hydrogen bonds in the wild and mutant structures. This study throws light on the possible effects of drug-resistant mutations on the large functionally important collective motions in biological systems.     

Inhibitory potency of flavonoid derivatives on influenza virus neuraminidase

The constant risk of emerging new influenza virus strains that are resistant to established inhibitors like oseltamivir leaves influenza neuraminidase (NA) a prominent target for drug design. The inhibitory activity of several flavonoid derivatives was experimentally tested in comparison to oseltamivir for the NA expressed by the seasonal influenza virus strains A/California/7/09 (A(H1N1)pdm09), A/Perth/16/09 (A(H3N2)), and B/Brisbane/60/08. IC₅₀ values of polyphenols confirmed moderate inhibition in the μM range. Structurally, the amount and site of glycosylation of tested flavonoids have no significant influence on their inhibitory potency. In a pharmacophore-based docking approach the structure–activity relationship was evaluated. Molecular dynamics simulations revealed highly flexible parts of the enzyme and the contribution of salt bridges to the structural stability of NA. The findings of this study elucidate the impact of flavonoids on viral neuraminidase activity and the analysis of their modes of action provide valuable information about the mechanism of NA inhibition.     

In silico prediction of drug resistance due to S247R mutation of Influenza H1N1 neuraminidase protein

We present here in silico studies on antiviral drug resistance due to a novel mutation of influenza A/H1N1 neuraminidase (NA) protein. Influenza A/H1N1 virus was responsible for a recent pandemic and is currently circulating among the seasonal influenza strains. M2 and NA are the two major viral proteins related to pathogenesis in humans and have been targeted for drug designing. Among them, NA is preferred because the ligand-binding site of NA is highly conserved between different strains of influenza virus. Different mutations of the NA active site residues leading to drug resistance or susceptibility of the virus were studied earlier. We report here a novel mutation (S247R) in the NA protein that was sequenced earlier from the nasopharyngeal swab from Sri Lanka and Thailand in the year 2009 and 2011, respectively. Another mutation (S247N) was already known to confer resistance to oseltamivir. We did a comparative study of these two mutations vis-a-vis the drug-sensitive wild type NA to understand the mechanism of drug resistance of S247N and to predict the probability of the novel S247R mutation to become resistant to the currently available drugs, oseltamivir and zanamivir. We performed molecular docking- and molecular dynamics-based analysis of both the mutant proteins and showed that mutation of S247R affects drug binding to the protein by positional displacement due to altered active site cavity architecture, which in turn reduces the affinity of the drug molecules to the NA active site. Our analysis shows that S247R may have high probability of being resistant.     

Computational studies of H5N1 influenza virus resistance to oseltamivir

Influenza A (H5N1) virus is one of the world's greatest pandemic threats. Neuraminidase (NA) inhibitors, oseltamivir and zanamivir, prevent the spread of influenza, but drug‐resistant viruses have reduced their effectiveness. Resistance depends on the binding properties of NA‐drug complexes. Key residue mutations within the active site of NA glycoproteins diminish binding, thereby resulting in drug resistance. We performed molecular simulations and calculations to characterize the mechanisms of H5N1 influenza virus resistance to oseltamivir and predict potential drug‐resistant mutations. We examined two resistant NA mutations, H274Y and N294S, and one non‐drug‐resistant mutation, E119G. Six‐nanosecond unrestrained molecular dynamic simulations with explicit solvent were performed using NA‐oseltamivir complexes containing either NA wild‐type H5N1 virus or a variant. MM_PBSA techniques were then used to rank the binding free energies of these complexes. Detailed analyses indicated that conformational change of E276 in the Pocket 1 region of NA is a key source of drug resistance in the H274Y mutant but not in the N294S mutant.     

In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.     

Analysis of oseltamivir resistance substitutions in influenza virus glycoprotein neuraminidase using a lentivirus-based surrogate assay system

Influenza A virus poses a great threat to global health, and oseltamivir (trade marked as Tamiflu), which targets influenza surface glycoprotein neuraminidase (NA), is used clinically as a major anti-influenza treatment. However, certain substitutions in NA can render an influenza virus resistant to this drug. In this study, using a lentiviral pseudotyping system, which alleviates the safety concerns of studying highly pathogenic influenza viruses such as avian influenza H5N1, that utilizes influenza surface glycoproteins (hemagglutinin or HA, and NA) and an HIV-core combined with a luciferase reporter gene as a surrogate assay, we first assessed the functionality of NA by measuring pseudovirion release in the absence or presence of oseltamivir. We demonstrated that oseltamivir displays a dose-dependent inhibition on NA activity. In contrast, a mutant NA (H274Y) is more resistant to oseltamivir treatment. In addition, the effects of several previously reported substitution NA mutants were examined as well. Our results demonstrate that this lentivirus-based pseudotyping system provides a quick, safe, and effective way to assess resistance to neuraminidase inhibitors. And we believe that as new mutations appear in influenza isolates, their impact on the effectiveness of current and future anti-NA can be quickly and reliably evaluated by this assay.     

Design,synthesis and biological evaluation of novel oseltamivir derivatives as potent neuraminidase inhibitors

Neuraminidase (NA) is one of the particular potential targets for novel antiviral therapy. In this work, a series of neuraminidase inhibitors with the cyclohexene scaffold were studied based upon the combination of 3D-QSAR, molecular docking, and molecular dynamics techniques. The results indicate that the built 3D-QSAR models yield reliable statistical information: the correlation coefficient (r²) and cross-validation coefficient (q²) of CoMFA (comparative molecular field analysis) are 0.992 and 0.819; the r² and q² of CoMSIA (comparative molecular similarity analysis) are 0.992 and 0.863, respectively. Molecular docking and MD simulations were conducted to confirm the detailed binding mode of enzyme-inhibitor system. The new NA inhibitors had been designed, synthesized, and their inhibitory activities against group-1 neuraminidase were determined. One agent displayed excellent neuraminidase inhibition, with IC₅₀ value of 39.6?μM against NA, while IC₅₀ value for oseltamivir is 61.1?μM. This compound may be further investigated for the treatment of infection by the new type influenza virus.     

The inhibitory performance of flavonoid cyanidin-3-sambubiocide against H274Y mutation in H1N1 influenza virus

Oseltamivir (Tamiflu) is the most accepted antiviral drug that targets the neuraminidase (NA) protein to inhibit the viral release from the host cell. Few H1N1 influenza strains with the H274Y mutation creates drug resistance to oseltamivir. In this study, we report that flavonoid cyanidin-3-sambubiocide (C3S) compound acts as a potential inhibitor against H274Y mutation. The drug resistance mechanism and inhibitory activity of C3S and oseltamivir against wild-type (WT) and H274Y mutant-type (MT) have been studied and compared based on the results of molecular docking, molecular dynamics, and quantum chemical methods. Oseltamivir has been found less binding affinity with MT. C3S has more binding affinity with WT and MT proteins. From the dynamical study, the 150th loop of the MT protein has found more deformation than WT. A single H274Y mutation induces the conformational changes in the 150th loop which leads to produce more resistance to oseltamivir. The 150th cavity is more attractive target for C3S to stop the conformational changes in the MT, than 430th cavity of NA protein. The C3S is stabilized with MT by more number of hydrogen bonds than oseltamivir. The electrostatic interaction energy shows a stronger C3S binding with MT and this compound may be more effective against oseltamivir-resistant virus strains.     

Oseltamivir-resistant influenza A viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol

Influenza viruses resistant to the neuraminidase (NA) inhibitor oseltamivir arise under drug selection pressure both in vitro and in vivo. Several mutations in the active site of the viral NA are known to confer relative resistance to oseltamivir, and influenza viruses with certain oseltamivir resistance mutations have been shown to transmit efficiently among cocaged ferrets. However, it is not known whether NA mutations alter aerosol transmission of drug-resistant influenza virus. Here, we demonstrate that recombinant human influenza A/H3N2 viruses without and with oseltamivir resistance mutations (in which NA carries the mutation E119V or the double mutations E119V I222V) have similar in ovo growth kinetics and infectivity in guinea pigs. These viruses also transmit efficiently by the contact route among cocaged guinea pigs, as in the ferret model. However, in an aerosol transmission model, in which guinea pigs are caged separately, the oseltamivir-resistant viruses transmit poorly or not at all; in contrast, the oseltamivir-sensitive virus transmits efficiently even in the absence of direct contact. The present results suggest that oseltamivir resistance mutations reduce aerosol transmission of influenza virus, which could have implications for public health measures taken in the event of an influenza pandemic.     

Detection of resistance mutations to antivirals oseltamivir and zanamivir in avian influenza A viruses isolated from wild birds

The neuraminidase (NA) inhibitors oseltamivir and zanamivir are the first-line of defense against potentially fatal variants of influenza A pandemic strains. However, if resistant virus strains start to arise easily or at a high frequency, a new anti-influenza strategy will be necessary. This study aimed to investigate if and to what extent NA inhibitor–resistant mutants exist in the wild population of influenza A viruses that inhabit wild birds. NA sequences of all NA subtypes available from 5490 avian, 379 swine and 122 environmental isolates were extracted from NCBI databases. In addition, a dataset containing 230 virus isolates from mallard collected at Ottenby Bird Observatory (Öland, Sweden) was analyzed. Isolated NA RNA fragments from Ottenby were transformed to cDNA by RT-PCR, which was followed by sequencing. The analysis of genotypic profiles for NAs from both data sets in regard to antiviral resistance mutations was performed using bioinformatics tools. All 6221 sequences were scanned for oseltamivir- (I117V, E119V, D198N, I222V, H274Y, R292K, N294S and I314V) and zanamivir-related mutations (V116A, R118K, E119G/A/D, Q136K, D151E, R152K, R224K, E276D, R292K and R371K). Of the sequences from the avian NCBI dataset, 132 (2.4%) carried at least one, or in two cases even two and three, NA inhibitor resistance mutations. Swine and environmental isolates from the same data set had 18 (4.75%) and one (0.82%) mutant, respectively, with at least one mutation. The Ottenby sequences carried at least one mutation in 15 cases (6.52%). Therefore, resistant strains were more frequently found in Ottenby samples than in NCBI data sets. However, it is still uncertain if these mutations are the result of natural variations in the viruses or if they are induced by the selective pressure of xenobiotics (e.g., oseltamivir, zanamivir).     

Pseudovirus-based neuraminidase inhibition assays reveal potential H5N1 drug-resistant mutations

The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. F or a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identifi ed to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.     

Molecular dynamics simulation of oseltamivir resistance in neuraminidase of avian influenza H5N1 virus

The outbreak of avian influenza virus H5N1 has raised a global concern because of its high virulence and mutation rate. Although two classes of antiviral drugs, M2 ion channel protein inhibitors and neuraminidase inhibitors, are expected to be important in controlling the early stages of a potential pandemic. Different strains of influenza viruses have differing degrees of resistance against the antivirals. In order to analyze the detailed information on the viral resistance, molecular dynamics simulations were carried out for the neuraminidase (NA) complex with oseltamivir. The carboxylate of Glu276 of H252Y NA faces toward the O-ethyl-propyl group of oesltamivir, Glu276 of wild-type NA adopts a conformation pointing away from the oesltamivir. τ2 and τ3 torsional angles fluctuation of the oesltamivir are relatively high for the H252Y mutant NA complex. In addition, there are fewer hydrogen bonds between the oesltamivir and H252Y mutation NA. The results show that H252Y mutation NA has high resistance against the drug.     

Binding mechanism of oseltamivir and influenza neuraminidase suggests perspectives for the design of new anti-influenza drugs

Oseltamivir is a widely used influenza virus neuraminidase (NA) inhibitor that prevents the release of new virus particles from host cells. However, oseltamivir-resistant strains have emerged, but effective drugs against them have not yet been developed. Elucidating the binding mechanisms between NA and oseltamivir may provide valuable information for the design of new drugs against NA mutants resistant to oseltamivir. Here, we conducted large-scale (353.4 μs) free-binding molecular dynamics simulations, together with a Markov State Model and an importance-sampling algorithm, to reveal the binding process of oseltamivir and NA. Ten metastable states and five major binding pathways were identified that validated and complemented previously discovered binding pathways, including the hypothesis that oseltamivir can be transferred from the secondary sialic acid binding site to the catalytic site. The discovery of multiple new metastable states, especially the stable bound state containing a water-mediated hydrogen bond between Arg118 and oseltamivir, may provide new insights into the improvement of NA inhibitors. We anticipated the findings presented here will facilitate the development of drugs capable of combating NA mutations.     

Inhibition of viral group-1 and group-2 neuraminidases by oseltamivir: A comparative structural analysis by the ScrewFit algorithm

The viral surface glycoprotein neuraminidase (NA) allows the influenza virus penetration and the egress of virions. NAs are classified as A, B, and C. Type-A NAs from influenza virus are subdivided into two phylogenetically distinct families, group-1 and group-2. NA inhibition by oseltamivir represents a therapeutic approach against the avian influenza virus H5N1. Here, structural bases for oseltamivir recognition by group-1 NA1, NA8 and group-2 NA9 are highlighted by the ScrewFit algorithm for quantitative structure comparison. Oseltamivir binding to NA1 and NA8 affects the geometry of Glu119 and of regions Arg130-Ser160, Val240-Gly260, and Asp330-Glu382, leading to multiple NA conformations. Additionally, although NA1 and NA9 share almost the same oseltamivir-bound final conformation, they show some relevant differences as suggested by the ScrewFit algorithm. These results indicate that the design of new NA inhibitors should take into account these family-specific effects induced on the whole structure of NAs.     

Efficacy of the New Neuraminidase Inhibitor CS-8958 against H5N1 Influenza Viruses

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 µg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants.     

Effects of Clinacanthus siamensis leaf extract on influenza virus infection

Ethanolic extracts of 20 medicinal plants were screened for influenza virus NA inhibition and in vitro antiviral activities using MDCK cells in an MTT assay. The vaccine proteins of influenza virus A/New Caledonia/20/99 (H1N1), mouse-adapted influenza virus A/Guizhou/54/89 (A/G)(H3N2) and mouse-adapted influenza virus B/Ibaraki/2/85 (B/I) were used in the NA inhibition assay, and mouse-adapted influenza viruses A/PR/8/34 (H1N1), A/G and B/I were used in the in vitro antiviral assay. The results of the in vitro antiviral assay indicated that the A/G virus was the most susceptible and an extract of the leaf of CS possessed the highest in vitro anti-A/G virus activity (41.98%). Therefore, the A/G virus and the CS extract were selected for studying in vivo anti-influenza virus activity. BALB/c mice were treated with CS extract (100 mg/kg per day, 5 times) orally from 4 hr before to 4 days after infection. CS extract elicited significant production of anti-influenza virus IgG₁ antibody in BAW and increased mouse weight compared to oseltamivir (0.1 mg/kg per day) on day 19 or water on days 17–19 of infection. Moreover, CS extract produced a higher anti-influenza virus IgA antibody level in BAW compared to oseltamivir, and a tendency towards an increase in anti-influenza virus IgA compared to water was shown. The results suggest that CS extract has a protective effect against influenza virus infection.     

Mass Spectrometry-Based Comparative Sequence Analysis for the Genetic Monitoring of Influenza A(H1N1)pdm09 Virus

The pandemic influenza A (H1N1) 2009 virus (pH1N1) contains novel gene segments of zoonotic origin that lack virulence and antiviral resistance markers. We aimed to evaluate the applicability and accuracy of mass spectrometry-based comparative sequence analysis (MSCSA) to detect genetic mutations associated with increased virulence or antiviral resistance in pH1N1. During the 2009 H1N1 pandemic, routine surveillance specimens and clinical antiviral resistance monitoring specimens were analyzed. Routine surveillance specimens obtained from 70 patients with pH1N1 infection were evaluated for mutations associated with increased virulence (PB1-F2, PB2 and NS1 genes) or antiviral resistance (neuraminidase gene, NA) using MSCSA and Sanger sequencing. MSCSA and Sanger sequencing results revealed a high concordance (nucleotides >99%, SNPs ∼94%). Virulence or resistance markers were not detected in routine surveillance specimens: all identified SNPs encoded for silent mutations or non-relevant amino acid substitutions. In a second study population, the presence of H275Y oseltamivir resistant virus was identified by real-time PCR in 19 of 35 clinical antiviral resistance monitoring specimens obtained from 4 immunocompromised patients with ≥14 days prolonged pH1N1 excretion. MSCSA detected H275Y in 24% (4/19) of positive specimens and Sanger sequencing in 89% (17/19). MSCSA only detected H275Y when the mutation was dominant in the analyzed specimens. In conclusion, MSCSA may be used as a rapid screening tool during molecular surveillance of pH1N1. The low sensitivity for the detection of H275Y mutation in mixed viral populations suggests that MSCSA is not suitable for antiviral resistance monitoring in the clinical setting.