Drug-resistant Influenza Strains Research Articles

Chemoenzymatic synthesis of sialyl-α2,3-lactoside–functionalized BSA conjugate inhibits influenza infection

Influenza remains a global public health threat, and the development of new antivirals is crucial to combat emerging drug-resistant influenza strains. In this study, we report the synthesis and evaluation of a sialyl lactosyl (TS)–bovine serum albumin (BSA) conjugate as a potential multivalent inhibitor of the influenza virus. The key trisaccharide component, TS, was efficiently prepared via a chemoenzymatic approach, followed by conjugation to dibenzocyclooctyne-modified BSA via a strain-promoted azide-alkyne cycloaddition reaction. Biophysical and biochemical assays, including surface plasmon resonance, isothermal titration calorimetry, hemagglutination inhibition, and neuraminidase inhibition, demonstrated the strong binding affinity of TS-BSA to the hemagglutinin (HA) and neuraminidase (NA) proteins of the influenza virus as well as intact virion particles. Notably, TS-BSA exhibited potent inhibitory activity against viral entry and release, preventing cytopathic effects in cell culture. This multivalent presentation strategy highlights the potential of glycocluster-based antivirals for combating influenza and other drug-resistant viral strains.

A novel compound to overcome influenza drug resistance in endonuclease inhibitors.

Influenza is a seasonal respiratory illness that kills hundreds of thousands of people every year. Currently, neuraminidase inhibitors and endonuclease inhibitors are used in antiviral therapy. However, both drug types have encountered drug-resistant influenza strains in the human body. Fortunately, there is currently no resistance to endonuclease inhibitors in wild strains of influenza. We obtained the molecules with endonuclease inhibitor activity independent of the existing drug-resistant strains through computer-aided drug design, and we hope the obtained results can lay a theoretical foundation for the development of high-activity endonucleases. Combining a traditional fragment-based drug discovery approach with AI-directed fragment growth, we selected and designed a compound that achieved antiviral activity on drug-resistant strains by avoiding mutable residues and drug-resistant residues. We predicted the related properties using an ADMET model. Finally, we obtained a compound similar to baloxavir in terms of binding free energy but not affected by baloxavir resistance.

Antiviral activities of plant-derived indole and β-carboline alkaloids against human and avian influenza viruses

The persistent evolution of drug-resistant influenza strains represents a global concern. The innovation of new treatment approaches through drug screening strategies and investigating the antiviral potential of bioactive natural-based chemicals may address the issue. Herein, we screened the anti-influenza efficacy of some biologically active indole and β-carboline (βC) indole alkaloids against two different influenza A viruses (IAV) with varied host range ranges; seasonal influenza A/Egypt/NRC098/2019(H1N1) and avian influenza A/chicken/Egypt/N12640A/2016(H5N1). All compounds were first assessed for their half-maximal cytotoxic concentration (CC50) in MDCK cells and half-maximal inhibitory concentrations (IC50) against influenza A/H5N1. Intriguingly, Strychnine sulfate, Harmalol, Harmane, and Harmaline showed robust anti-H5N1 activities with IC50 values of 11.85, 0.02, 0.023, and 3.42 µg/ml, respectively, as compared to zanamivir and amantadine as control drugs (IC50 = 0.079 µg/ml and 17.59 µg/ml, respectively). The efficacy of the predefined phytochemicals was further confirmed against influenza A/H1N1 and they displayed potent anti-H1N1 activities compared to reference drugs. Based on SI values, the highly promising compounds were then evaluated for antiviral efficacy through plaque reduction assay and consistently they revealed high viral inhibition percentages at non-toxic concentrations. By studying the modes of antiviral action, Harmane and Harmalol could suppress viral infection via interfering mainly with the viral replication of the influenza A/H5N1 virus, whilst Harmaline exhibited a viricidal effect against the influenza A/H5N1 virus. Whereas, Strychnine sulfate elucidated its anti-influenza potency by interfering with viral adsorption into MDCK cells. Consistently, chemoinformatic studies showed that all studied phytochemicals illustrated HB formations with essential peptide cleft through the NH of indole moiety. Among active alkaloids, harmalol displayed the best lipophilicity metrics including ligand efficiency (LE) and ligand lipophilic efficiency (LLE) for both viruses. Compounds geometry and their ability to participate in HB formation are very crucial.

2D-QSAR, 3D-QSAR, molecular docking and ADMET prediction studies of some novel 2-((1H-indol-3-yl)thio)-N-phenyl-acetamide derivatives as anti-influenza A virus

ABSTRACT Due to the emergence of drug-resistant strains of influenza A virus (IAV) in recent times, the need to search and discover more potent anti-IAV inhibitors is of great interest, especially with the devastating COVID-19 pandemic. The present research applied 2D-QSAR, 3D-QSAR, molecular docking, and ADMET predictions on some novel analogs of 2-((1 H-indol-3-yl)thio)-N-phenyl-acetamide as IAV inhibitors. The 2D-QSAR modeling results revealed GFA-MLR (=0.8861, q2 = 0.7864) and GFA-ANN (=0.8980, q2 = 0.8884) models with the most relevant descriptors for predicting the anti-IAV responses of the compounds, which have passed the benchmarks for accepting QSAR models. The 3D-QSAR modeling results suggested CoMFA_SE (=0.925, q2 = 0.59) and CoMSIA_EAD (=0.929, q2 = 0.767) models for good and reliable activity predictions. The molecular docking of the compounds with the active site of neuraminidase (NA) receptor theoretically confirms their resilient potency. The compounds mostly formed H-bond and hydrophobic interactions with key residues, such as ARG118, ASP151, GLU119, TRP179, ARG293 and PRO431 that triggered the catalytic reaction for the NA inhibition. However, compounds 16 and 21 were identified as lead compounds in the in-silico search for more potent candidates. The outcome of this study set a course for the in-silico design and search of potential candidates for influenza therapy.

An anti-influenza A virus microbial metabolite acts by degrading viral endonuclease PA

The emergence of new highly pathogenic and drug-resistant influenza strains urges the development of novel therapeutics for influenza A virus (IAV). Here, we report the discovery of an anti-IAV microbial metabolite called APL-16-5 that was originally isolated from the plant endophytic fungus Aspergillus sp. CPCC 400735. APL-16-5 binds to both the E3 ligase TRIM25 and IAV polymerase subunit PA, leading to TRIM25 ubiquitination of PA and subsequent degradation of PA in the proteasome. This mode of action conforms to that of a proteolysis targeting chimera which employs the cellular ubiquitin-proteasome machinery to chemically induce the degradation of target proteins. Importantly, APL-16-5 potently inhibits IAV and protects mice from lethal IAV infection. Therefore, we have identified a natural microbial metabolite with potent in vivo anti-IAV activity and the potential of becoming a new IAV therapeutic. The antiviral mechanism of APL-16-5 opens the possibility of improving its anti-IAV potency and specificity by adjusting its affinity for TRIM25 and viral PA protein through medicinal chemistry.

Brevilin A, a Sesquiterpene Lactone, Inhibits the Replication of Influenza A Virus In Vitro and In Vivo.

With the emergence of drug-resistant strains of influenza A viruses (IAV), new antivirals are needed to supplement the existing counter measures against IAV infection. We have previously shown that brevilin A, a sesquiterpene lactone isolated from C. minima, suppresses the infection of influenza A/PR/8/34 (H1N1) in vitro. Here, we further investigate the antiviral activity and mode of action of brevilin A against different IAV subtypes. Brevilin A inhibited the replication of influenza A H1N1, H3N2, and H9N2 viruses in vitro. The suppression effect of brevilin A was observed as early as 4–8 hours post infection (hpi). Furthermore, we determined that brevilin A inhibited viral replication in three aspects, including viral RNA (vRNA) synthesis, expression of viral mRNA, and protein encoded from the M and NS segments, and nuclear export of viral ribonucleoproteins (vRNPs). The anti-IAV activity of brevilin A was further confirmed in mice. A delayed time-to-death with 50% surviving up to 14 days post infection was obtained with brevilin A (at a dose of 25 mg/kg) treated animals compared to the control cohorts. Together, these results are encouraging for the exploration of sesquiterpene lactones with similar structure to brevilin A as potential anti-influenza therapies.

Structural modification on rupestonic acid leads to highly potent inhibitors against influenza virus.

Influenza viruses are responsible for seasonal epidemics and occasional pandemics, which cause significant morbidity and mortality. Although several drugs (adamantanes and neuraminidase inhibitors) are available in the market, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs. Artemisia rupestris L. is a folk medicine used to treat cold. In this paper, we structurally modified rupestonic acid, a bioactive component of A. rupestris, to synthesize a series of 2-substituted rupestonic acid methyl esters (3a-3o). Their structures were fully characterized by 1H NMR, 13C NMR, HRMS spectra. Among them, compounds 3b and 3c exhibited potent activities against influenza H1N1 with micromolar IC50 values and might serve as new lead compounds for the treatment of influenza.

Applications of Influenza Viral Kinetic Modeling in Drug Development

Seasonal influenza epidemics continue to cause significant morbidity and mortality, especially in high-risk subpopulations. The rise of drug-resistant influenza strains and high rates of vaccine mismatches drives the search for new safe and effective antiviral drugs. The purpose of this review was to summarize how mathematical models―viral kinetic models, in particular―have advanced our understanding of influenza biology and antiviral pharmacology and to explore how these models may be further developed in the future. Viral kinetic models that use a population approach have been helpful in elucidating the major sources of intra- and inter-individual variability. Thus, these models can help explain why some influenza patients become much sicker than others. Linking viral kinetic models to pharmacokinetic/pharmacodynamic models has enabled quantification of drug effects based on their mechanism of action and in silico evaluation of the efficacy of drug combinations. Viral kinetic models have also been applied to optimizing the design of clinical trials. Future directions for viral kinetic models include coupling to epidemiological models to assess the effectiveness of various public health treatment strategies. These models can also inform health economic decision-making. Since the 1970s, the field of viral kinetic modeling of influenza has made great advances in informing our knowledge of this infectious disease. This tool is being coupled to other types of modeling approaches and will continue to be a critical weapon in our infectious disease armamentarium.

Antiviral Activity of Aspalathus linearis against Human Influenza Virus

Influenza A viruses are responsible for annual epidemics and occasional pandemics, which cause significant morbidity and mortality. The limited protection offered by influenza vaccination, and the emergence of drug-resistant influenza strains, highlight the urgent need for the development of novel anti-influenza drugs. However, the search for antiviral substances from the library of low molecular weight chemical compounds is limited. Thus, because of their natural diversity and accessibility, plants or plant-derived materials are rapidly becoming valuable sources for the discovery and development of new antiviral drugs. In this study, crude extracts of Aspalathus linearis, a plant reported to have anti-HIV activity, were evaluated in vitro for their activity against the influenza A virus Of the extracts tested, an alkaline extract of Aspalathus linearis demonstrated the strongest inhibition against influenza A virus and could also inhibit different.types of influenza viruses, including Oseltamivir-resistant influenza viruses A and B. Our time course of addition studies indicated that the alkaline extract of Aspalathus linearis exerts its antiviral effect predominantly during the late stages of the influenza virus replication process.

Aliphatic and alicyclic camphor imines as effective inhibitors of influenza virus H1N1

A series of camphor derived imines was synthesised and evaluated in vitro for antiviral activity. Theoretical evaluations of ADME properties were also carried out. Most of these compounds exhibited significant activity against the drug-resistant strains of influenza A virus. Especially, compounds 2 (SI = 632) and 3 (SI = 417) presented high inhibition against influenza subtypes A/Puerto Rico/8/34 and A/California/07/09 of H1N1pdm09. Analysis of the structure-activity relationship showed that the activity was strongly dependent on the length of the aliphatic chain: derivatives with a shorter chain possessed higher activity, while the suppressing action of compounds with long aliphatic chains was lower.

Conformational Response of Influenza A M2 Transmembrane Domain to Amantadine Drug Binding at Low pH (pH 5.5).

The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21−49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21−49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21−49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21−49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L's ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5–8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21−49 through increased motional ordering. In contrast to wild-type M2TMD21−49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain drug-resistant influenza strains. Thus, the inhibited M2TMD21−49 channel is a stable tetramer with a closed C-terminal exit pore. This work is aimed at contributing to the development of structure-based anti-influenza pharmaceuticals.

Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and Pyrimidine C-Nucleosides as Favipiravir (T-705) Analogues.

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.

Perspective of Use of Antiviral Peptides against Influenza Virus.

The threat of a worldwide influenza pandemic has greatly increased over the past decade with the emergence of highly virulent avian influenza strains. The increased frequency of drug-resistant influenza strains against currently available antiviral drugs requires urgent development of new strategies for antiviral therapy, too. The research in the field of therapeutic peptides began to develop extensively in the second half of the 20th century. Since then, the mechanisms of action for several peptides and their antiviral prospect received large attention due to the global threat posed by viruses. Here, we discussed the therapeutic properties of peptides used in influenza treatment. Peptides with antiviral activity against influenza can be divided into three main groups. First, entry blocker peptides such as a Flupep that interact with influenza hemagglutinin, block its binding to host cells and prevent viral fusion. Second, several peptides display virucidal activity, disrupting viral envelopes, e.g., Melittin. Finally, a third set of peptides interacts with the viral polymerase complex and act as viral replication inhibitors such as PB1 derived peptides. Here, we present a review of the current literature describing the antiviral activity, mechanism and future therapeutic potential of these influenza antiviral peptides.

A virtual screening approach for identifying plants with anti H5N1 neuraminidase activity.

Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 μM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources.

Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus

Influenza (flu) pandemics have exhibited a great threat to human health throughout history. With the emergence of drug-resistant strains of influenza A virus (IAV), it is necessary to look for new agents for treatment and transmission prevention of the flu. Defensins are small (2–6 kDa) cationic peptides known for their broad-spectrum antimicrobial activity. Beta-defensins (β-defensins) are mainly produced by barrier epithelial cells and play an important role in attacking microbe invasion by epithelium. In this study, we focused on the anti-influenza A virus activity of mouse β-defensin 1 (mBD1) and β defensin-3 (mBD3) by synthesizing their fusion peptide with standard recombinant methods. The eukaryotic expression vectors pcDNA3.1(+)/mBD1-mBD3 were constructed successfully by overlap-PCR and transfected into Madin-Darby canine kidney (MDCK) cells. The MDCK cells transfected by pcDNA3.1(+)/mBD1-mBD3 were obtained by G418 screening, and the mBD1-mBD3 stable expression pattern was confirmed in MDCK cells by RT-PCR and immunofluorescence assay. The acquired stable transfected MDCK cells were infected with IAV (A/PR/8/34, H1N1, 0.1 MOI) subsequently and the virus titers in cell culture supernatants were analyzed by TCID50 72 h later. The TCID50 titer of the experimental group was clearly lower than that of the control group (p < 0.001). Furthermore, BALB/C mice were injected with liposome-encapsulated pcDNA3.1(+)/mBD1-mBD3 through muscle and then challenged with the A/PR/8/34 virus. Results showed the survival rate of 100% and lung index inhibitory rate of 32.6% in pcDNA3.1(+)/mBD1-mBD3group; the TCID50 titer of lung homogenates was clearly lower than that of the control group (p < 0.001). This study demonstrates that mBD1-mBD3 expressed by the recombinant plasmid pcDNA3.1(+)/mBD1-mBD3 could inhibit influenza A virus replication both in vitro and in vivo. These observations suggested that the recombinant mBD1-mBD3 might be developed into an agent for influenza prevention and treatment.

Detection of Oseltamivir-Resistant Pandemic Influenza A(H1N1)pdm2009 in Brazil: Can Community Transmission Be Ruled Out?

Although surveillance efforts that monitor the emergence of drug-resistant strains of influenza are critical, systematic analysis is overlooked in most developing countries. We report on the occurrence of strains of pandemic influenza A(H1N1)pdm09 with resistance and decreased susceptibility to oseltamivir (OST) in Brazil in 2009, 2011 and 2012. We found 7 mutant viruses, 2 with the mutation S247N and other 5 with the mutation H275Y. Most of these viruses were from samples concentrated in the southern region of Brazil. Some of these resistant viruses were detected prior to the initiation of OST treatment, suggesting that community transmission of mutant viruses may exist. Moreover, we show that one of these OST-resistant (H275Y) strains of A(H1N1)pdm09 was discovered in the tri-border region between Brazil, Argentina and Paraguay, highlighting that this strain could also be found in other Latin American countries. Our findings reinforce the importance of enhanced antiviral resistance surveillance in Brazil and in other Latin American countries to confirm or rule out the community transmission of OST-resistant strains of A(H1N1)pdm09.

Crystallographic fragment screening and structure-based optimization yields a new class of influenza endonuclease inhibitors.

Seasonal and pandemic influenza viruses continue to be a leading global health concern. Emerging resistance to the current drugs and the variable efficacy of vaccines underscore the need for developing new flu drugs that will be broadly effective against wild-type and drug-resistant influenza strains. Here, we report the discovery and development of a class of inhibitors targeting the cap-snatching endonuclease activity of the viral polymerase. A high-resolution crystal form of pandemic 2009 H1N1 influenza polymerase acidic protein N-terminal endonuclease domain (PAN) was engineered and used for fragment screening leading to the identification of new chemical scaffolds binding to the PAN active site cleft. During the course of screening, binding of a third metal ion that is potentially relevant to endonuclease activity was detected in the active site cleft of PAN in the presence of a fragment. Using structure-based optimization, we developed a highly potent hydroxypyridinone series of compounds from a fragment hit that defines a new mode of chelation to the active site metal ions. A compound from the series demonstrating promising enzymatic inhibition in a fluorescence-based enzyme assay with an IC50 value of 11 nM was found to have an antiviral activity (EC50) of 11 μM against PR8 H1N1 influenza A in MDCK cells.

Conjugation to Polymeric Chains of Influenza Drugs Targeting M2 Ion Channels Partially Restores Inhibition of Drug-Resistant Mutants

By attaching multiple copies of the influenza M2 ion channel inhibitors amantadine (1) and rimantadine (2) to polymeric chains, we endeavored to recover their potency in inhibiting drug-resistant influenza viruses. Depending on loading densities, as well as the nature of the drug, the polymer, and the spacer arm, polymer-conjugated drugs were up to 30-fold more potent inhibitors of drug-resistant strains than their monomeric parents. In particular, a 20% loading density and a short linker group on the negatively charged poly-l-glutamate resulted in one of the most potent inhibitors for 2's conjugates against drug-resistant influenza strains. Although full recovery of the inhibitory action against drug-resistant strains was not achieved, this study may be a step toward salvaging anti-influenza drugs that are no longer effective.

Theoretical Studies on the Susceptibility of Oseltamivir against Variants of 2009 A/H1N1 Influenza Neuraminidase

The outbreak and high speed global spread of the new strain of influenza A/H1N1 virus in 2009 posed a serious threat to global health. It is more likely that drug-resistant influenza strains will arise after the extensive use of anti-influenza drugs. Consequently, the identification of the potential resistant sites for drugs in advance and the understanding of the corresponding molecular mechanisms that cause drug resistance are quite important in the design of new drug candidates with better potency to combat drug resistance. Here, we performed molecular simulations to evaluate the potency of oseltamivir to combat drug resistance caused by the mutations in 2009 A/H1N1 neuraminidase (NA). We examined three representative drug-resistant mutations in NA, consisting of H274Y, N294S, and Y252H. First, a theoretical structure of A/H1N1 NA in complex with oseltamivir was constructed using homology modeling. Then, molecular dynamics (MD) simulations, molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculations, and MM/GBSA free energy decomposition were used to characterize the binding of oseltamivir with the wild type (WT) and three mutated NAs. Our predictions show that N294S and H274Y, two popular drug-resistant mutations in different variants of NA, still cause significant resistance to oseltamivir. However, the Y252H mutation does not impair the interactions between oseltamivir and A/H1N1 NA. An examination of individual energy components shows that the loss of polar interactions is the key source for the resistance of the studied mutations to oseltamivir. Moreover, free energy decomposition analysis and structural analysis reveal that the N294S or H274Y mutation triggers the large-scale conformational changes of the binding pocket and then impairs the affinity of oseltamivir. We expect that our results will be useful for the rational design of NA inhibitors with high potency against drug-resistant A/H1N1 mutants.

Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an example

BackgroundPositive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance.ResultsUsing influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3' ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor (NAI) resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays.ConclusionThis approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.