Triple Combination of Amantadine,Ribavirin, and Oseltamivir Is Highly Active and Synergistic against Drug Resistant Influenza Virus Strains In Vitro

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21^st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza.     

Efficacy of combined therapy with amantadine, oseltamivir, and ribavirin in vivo against susceptible and amantadine-resistant influenza A viruses

The limited efficacy of existing antiviral therapies for influenza--coupled with widespread baseline antiviral resistance--highlights the urgent need for more effective therapy. We describe a triple combination antiviral drug (TCAD) regimen composed of amantadine, oseltamivir, and ribavirin that is highly efficacious at reducing mortality and weight loss in mouse models of influenza infection. TCAD therapy was superior to dual and single drug regimens in mice infected with drug-susceptible, low pathogenic A/H5N1 (A/Duck/MN/1525/81) and amantadine-resistant 2009 A/H1N1 influenza (A/California/04/09). Treatment with TCAD afforded >90% survival in mice infected with both viruses, whereas treatment with dual and single drug regimens resulted in 0% to 60% survival. Importantly, amantadine had no activity as monotherapy against the amantadine-resistant virus, but demonstrated dose-dependent protection in combination with oseltamivir and ribavirin, indicative that amantadine's activity had been restored in the context of TCAD therapy. Furthermore, TCAD therapy provided survival benefit when treatment was delayed until 72 hours post-infection, whereas oseltamivir monotherapy was not protective after 24 hours post-infection. These findings demonstrate in vivo efficacy of TCAD therapy and confirm previous reports of the synergy and broad spectrum activity of TCAD therapy against susceptible and resistant influenza strains in vitro.     

Characterization of inhibition of M2 ion channel activity by BL-1743, an inhibitor of influenza A virus.

The influenza A virus M2 integral membrane protein has ion channel activity that can be inhibited by the antiviral drug amantadine. Recently, a spirene-containing compound, BL-1743 (2-[3-azaspiro (5,5)undecanol]-2-imidazoline), that inhibits influenza virus growth was identified (S. Kurtz, G. Lao, K. M. Hahnenberger, C. Brooks, O. Gecha, K. Ingalls, K.-I. Numata, and M. Krystal, Antimicrob. Agents Chemother. 39:2204-2209, 1995). We have examined the ability of BL-1743 to inhibit the M2 ion channel when expressed in oocytes of Xenopus laevis. BL-1743 inhibition is complete as far as can be measured by electrophysiological methods and is reversible, with a reverse reaction rate constant of 4.0 x 10(-3) s(-1). In contrast, amantadine inhibition is irreversible within the time frame of the experiment. However, BL-1743 inhibition and amantadine inhibition have similar properties. The majority of isolated influenza viruses resistant to BL-1743 are also amantadine resistant. In addition, all known amino acid changes which result in amantadine resistance also confer BL-1743 resistance. However, one BL-1743-resistant virus isolated, designated M2-I35T, contained the change Ile-35-->Thr. This virus is >70-fold more resistant to BL-1743 and only 10-fold more resistant to amantadine than the wild-type virus. When the ion channel activity of M2-I35T was examined in oocytes, it was found that M2-I35T is BL-1743 resistant but is reversibly inhibited by amantadine. These findings suggest that these two drugs interact differently with the M2 protein transmembrane pore region.     

Incidence of amantadine-resistant influenza A viruses in sentinel surveillance sites and nursing homes in Niigata, Japan

We surveyed the incidence of amantadine-resistant influenza A viruses both at sentinel surveillance sites and at nursing homes, and verified their types of change by partial nucleotide sequence analysis of the M2 protein. Fifty-five influenza A viruses from 27 sentinel surveillance sites during six influenza seasons from 1993 to 1999, and 26 influenza A viruses from 5 nursing homes from 1996 to 1999 were examined for susceptibility to the drug by virus titration in the presence or absence of amantadine. While amantadine-resistant viruses were not found in sentinel surveillance sites, a high frequency of resistance (8/26, 30.8%) in nursing homes was observed. Resistant viruses can occur quickly and be transmitted when used in an outbreak situation at nursing homes, where amantadine is used either for neurologic indications or for influenza treatment. Eight resistant viruses had a single amino acid change of the M2 protein at residue 30 or 31. In vitro, all 11 sensitive viruses turned resistant after 3 or 5 passages in the presence of 2 microg/ml amantadine, and they showed an amino acid change at residue 27, 30, or 31. The predominant amino acid substitution in the M2 protein of resistant viruses is Ser-31-Asp (a change at 31, serine to asparagine). The results indicate that a monitoring system for amantadine-resistant influenza viruses should be established without delay in Japan.     

Solution NMR structure of the V27A drug resistant mutant of influenza A M2 channel

The M2 protein of influenza A virus forms a proton-selective channel that is required for viral replication. It is the target of the anti-influenza drugs, amantadine and rimantadine. Widespread drug resistant mutants, however, has greatly compromised the effectiveness of these drugs. Here, we report the solution NMR structure of the highly pathogenic, drug resistant mutant V27A. The structure reveals subtle structural differences from wildtype that maybe linked to drug resistance. The V27A mutation significantly decreases hydrophobic packing between the N-terminal ends of the transmembrane helices, which explains the looser, more dynamic tetrameric assembly. The weakened channel assembly can resist drug binding either by destabilizing the rimantadine-binding pocket at Asp44, in the case of the allosteric inhibition model, or by reducing hydrophobic contacts with amantadine in the pore, in the case of the pore-blocking model. Moreover, the V27A structure shows a substantially increased channel opening at the N-terminal end, which may explain the faster proton conduction observed for this mutant. Furthermore, due to the high quality NMR data recorded for the V27A mutant, we were able to determine the structured region connecting the channel domain to the C-terminal amphipathic helices that was not determined in the wildtype structure. The new structural data show that the amphipathic helices are packed much more closely to the channel domain and provide new insights into the proton transfer pathway.     

In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675

With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.     

Characterization of a human immunodeficiency virus type 1 variant with reduced sensitivity to an aminodiol protease inhibitor.

Development of viral resistance to the aminodiol human immunodeficiency virus (HIV) protease inhibitor BMS 186,318 was studied by serial passage of HIV type 1 RF in MT-2 cells in the presence of increasing concentrations of compound. After 11 passages, an HIV variant that showed a 15-fold increase in 50% effective dose emerged. This HIV variant displays low-level cross-resistance to the C2 symmetric inhibitor A-77003 but remains sensitive to the protease inhibitors Ro 31-8959 and SC52151. Genetic analysis of the protease gene from a drug-resistant variant revealed an Ala-to-Thr change at amino acid residue 71 (A71T) and a Val-to-Ala change at residue 82 (V82A). To determine the effects of these mutations on protease and virus drug susceptibility, recombinant protease and proviral HIV type 1 clones containing the single mutations A71T and V82A or double mutation A71T/V82A were constructed. Subsequent drug sensitivity assays on the mutant proteases and viruses indicated that the V82A substitution was responsible for most of the resistance observed. Further genotypic analysis of the protease genes from earlier passages of virus indicated that the A71T mutation emerged prior to the V82A change. Finally, the level of resistance did not increase following continued passage in increasing concentrations of drug, and the resistant virus retained its drug susceptibility phenotype 34 days after drug withdrawal.     

焦磷酸测序技术在禽流感病毒金刚烷胺耐药性检测中的应用

流感病毒M2蛋白五个关键位点氨基酸残基(第26、27、30、31和34位)中的任何一个发生突变都会导致抗流感病毒药物中金刚烷胺抗药性的产生。本研究利用焦磷酸测序技术对94株不同亚型禽流感病毒金刚烷胺耐药性分子决定区进行了鉴定,并进行抗药性分析。结果表明94株禽流感病毒中有81株M2基因存在金刚烷胺耐药性的分子标签,其余的13株根据分子标签判断为对金刚烷胺敏感。耐药性的分子标记存在V27I和S31N两种突变形式,其中绝大多数为S31N。     

Antiviral resistance and the control of pandemic influenza: the roles of stochasticity, evolution and model details

Antiviral drugs, most notably the neuraminidase inhibitors, are an important component of control strategies aimed to prevent or limit any future influenza pandemic. The potential large-scale use of antiviral drugs brings with it the danger of drug resistance evolution. A number of recent studies have shown that the emergence of drug-resistant influenza could undermine the usefulness of antiviral drugs for the control of an epidemic or pandemic outbreak. While these studies have provided important insights, the inherently stochastic nature of resistance generation and spread, as well as the potential for ongoing evolution of the resistant strain have not been fully addressed. Here, we study a stochastic model of drug resistance emergence and consecutive evolution of the resistant strain in response to antiviral control during an influenza pandemic. We find that taking into consideration the ongoing evolution of the resistant strain does not increase the probability of resistance emergence; however, it increases the total number of infecteds if a resistant outbreak occurs. Our study further shows that taking stochasticity into account leads to results that can differ from deterministic models. Specifically, we find that rapid and strong control cannot only contain a drug sensitive outbreak, it can also prevent a resistant outbreak from occurring. We find that the best control strategy is early intervention heavily based on prophylaxis at a level that leads to outbreak containment. If containment is not possible, mitigation works best at intermediate levels of antiviral control. Finally, we show that the results are not very sensitive to the way resistance generation is modeled.     

Aureonitol,a Fungi-Derived Tetrahydrofuran,Inhibits Influenza Replication by Targeting Its Surface Glycoprotein Hemagglutinin

The influenza virus causes acute respiratory infections, leading to high morbidity and mortality in groups of patients at higher risk. Antiviral drugs represent the first line of defense against influenza, both for seasonal infections and pandemic outbreaks. Two main classes of drugs against influenza are in clinical use: M2-channel blockers and neuraminidase inhibitors. Nevertheless, because influenza strains that are resistant to these antivirals have been described, the search for novel compounds with different mechanisms of action is necessary. Here, we investigated the anti-influenza activity of a fungi-derived natural product, aureonitol. This compound inhibited influenza A and B virus replication. This compound was more effective against influenza A(H3N2), with an EC₅₀ of 100 nM. Aureonitol cytoxicity was also very low, with a CC₅₀ value of 1426 μM. Aureonitol inhibited influenza hemagglutination and, consequently, significantly impaired virus adsorption. Molecular modeling studies revealed that aureonitol docked in the sialic acid binding site of hemagglutinin, forming hydrogen bonds with highly conserved residues. Altogether, our results indicate that the chemical structure of aureonitol is promising for future anti-influenza drug design.     

Influenza A Virus M2 Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656-5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del(29-31)) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del(29-31) virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del(29-31) virus. We found that the M2-del(29-31) virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del(29-31) virus in 4 days after cocultivation of a 100:1 ratio of M2-del(29-31) virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.     

Hedging against Antiviral Resistance during the Next Influenza Pandemic Using Small Stockpiles of an Alternative Chemotherapy

Background

The effectiveness of single-drug antiviral interventions to reduce morbidity and mortality during the next influenza pandemic will be substantially weakened if transmissible strains emerge which are resistant to the stockpiled antiviral drugs. We developed a mathematical model to test the hypothesis that a small stockpile of a secondary antiviral drug could be used to mitigate the adverse consequences of the emergence of resistant strains.

Methods and Findings

We used a multistrain stochastic transmission model of influenza to show that the spread of antiviral resistance can be significantly reduced by deploying a small stockpile (1% population coverage) of a secondary drug during the early phase of local epidemics. We considered two strategies for the use of the secondary stockpile: early combination chemotherapy (ECC; individuals are treated with both drugs in combination while both are available); and sequential multidrug chemotherapy (SMC; individuals are treated only with the secondary drug until it is exhausted, then treated with the primary drug). We investigated all potentially important regions of unknown parameter space and found that both ECC and SMC reduced the cumulative attack rate (AR) and the resistant attack rate (RAR) unless the probability of emergence of resistance to the primary drug p_A was so low (less than 1 in 10,000) that resistance was unlikely to be a problem or so high (more than 1 in 20) that resistance emerged as soon as primary drug monotherapy began. For example, when the basic reproductive number was 1.8 and 40% of symptomatic individuals were treated with antivirals, AR and RAR were 67% and 38% under monotherapy if p _A = 0.01. If the probability of resistance emergence for the secondary drug was also 0.01, then SMC reduced AR and RAR to 57% and 2%. The effectiveness of ECC was similar if combination chemotherapy reduced the probabilities of resistance emergence by at least ten times. We extended our model using travel data between 105 large cities to investigate the robustness of these resistance-limiting strategies at a global scale. We found that as long as populations that were the main source of resistant strains employed these strategies (SMC or ECC), then those same strategies were also effective for populations far from the source even when some intermediate populations failed to control resistance. In essence, through the existence of many wild-type epidemics, the interconnectedness of the global network dampened the international spread of resistant strains.

Conclusions

Our results indicate that the augmentation of existing stockpiles of a single anti-influenza drug with smaller stockpiles of a second drug could be an effective and inexpensive epidemiological hedge against antiviral resistance if either SMC or ECC were used. Choosing between these strategies will require additional empirical studies. Specifically, the choice will depend on the safety of combination therapy and the synergistic effect of one antiviral in suppressing the emergence of resistance to the other antiviral when both are taken in combination.     

Multiple drug resistance inAerobacter aerogenes

Multiple drug resistance resulting from continued growth in various drugs in turn followed by serial subculture in the simultaneous presence of all the drugs has been studied. In this way strains ofA. aerogenes resistant, in considerable measure, to 3, 4 and 5 drugs have been obtained. Although the final state was always a compromise between the various new properties it was often a good and very effective compromise and for this reason kinetic properties of the cells such as growth rate had to be intensively followed. Tests for presence or absence of growth after a fixed interval of time would not have sufficed. At the 5-drug stage a good compromise was obtained when the agents used were streptomycin, sulphanilamide, chloramphenicol, ampicillin and 5-aminoacridine. When tretamine (triethylene melamine) replaced 5-aminoacridine as the fifth drug, the rate of growth in the various drugs both singly and in admixture was not as good.Continued subculture in sulphanilamide medium led to a condition of hypersensitivity to chloramphenicol. Ampicillin-resistant strains were more inhibited by 2-phenylethanol than the ampicillin-sensitive parent organism. Strains resistant to chloramphenicol were also resistant to tretamine and vice versa. Such cross-resistance did not occur with any of the other drugs at the concentrations used.     

Influenza virus M2 protein has ion channel activity.

The influenza virus M2 protein was expressed in Xenopus laevis oocytes and shown to have an associated ion channel activity selective for monovalent ions. The anti-influenza virus drug amantadine hydrochloride significantly attenuated the inward current induced by hyperpolarization of oocyte membranes. Mutations in the M2 membrane-spanning domain that confer viral resistance to amantadine produced currents that were resistant to the drug. Analysis of the currents of these altered M2 proteins suggests that the channel pore is formed by the transmembrane domain of the M2 protein. The wild-type M2 channel was found to be regulated by pH. The wild-type M2 ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection.     

应用焦磷酸测序技术快速检测猪流感病毒金刚烷胺耐药性的分子标签

【目的】本研究旨在通过焦磷酸测序技术对我国分离的H1N1、H3N2、H9N2等3种基因型的10株猪流感病毒分离株进行金刚烷胺耐药性鉴定。【方法】流感病毒M2蛋白5个关键位点氨基酸残基(第26、27、30、31和34位)中的任何一个发生突变会导致抗流感病毒药物中金刚烷胺抗药性的产生。本研究利用焦磷酸测序技术对2004-2008年国内分离的10株猪流感病毒M基因金刚烷胺耐药性分子决定区进行了鉴定,并进行抗药性分析。【结果】基于M2蛋白基因保守区序列建立的焦磷酸测序技术能用于国内猪流感病毒的快速检测,且具有较好的特异性和重复性。抗药性分析表明10株猪流感病毒国内分离株中5株H1N1分离株全部耐药,主要存在M2蛋白的V27T、V27I或S31N位点的突变,而4株H3N2和1株H9N2猪流感病毒分离株在M2蛋白5个关键位点上均未出现变异,表明其对金刚烷胺敏感。【结论】基于M基因的焦磷酸测序技术可以用于我国猪流感病毒金刚烷胺耐药性快速鉴定。     

HIV-1 Resistance to Maraviroc Conferred by a CD4 Binding Site Mutation in the Envelope Glycoprotein gp120

Maraviroc (MVC) is a CCR5 antagonist that inhibits HIV-1 entry by binding to the coreceptor and inducing structural alterations in the extracellular loops. In this study, we isolated MVC-resistant variants from an HIV-1 primary isolate that arose after 21 weeks of tissue culture passage in the presence of inhibitor. gp120 sequences from passage control and MVC-resistant cultures were cloned into NL4-3 via yeast-based recombination followed by sequencing and drug susceptibility testing. Using 140 clones, three mutations were linked to MVC resistance, but none appeared in the V3 loop as was the case with previous HIV-1 strains resistant to CCR5 antagonists. Rather, resistance was dependent upon a single mutation in the C4 region of gp120. Chimeric clones bearing this N425K mutation replicated at high MVC concentrations and displayed significant shifts in 50% inhibitory concentrations (IC₅₀s), characteristic of resistance to all other antiretroviral drugs but not typical of MVC resistance. Previous reports on MVC resistance describe an ability to use a drug-bound form of the receptor, leading to reduction in maximal drug inhibition. In contrast, our structural models on K425 gp120 suggest that this resistant mutation impacts CD4 interactions and highlights a novel pathway for MVC resistance.     

Comparison of spontaneous, UV-induced, and nitrosoguanidine-induced mutability to drug resistance in myxobacteria.

The UV survival curves of different strains of myxobacteria exhibited shoulders; in the case of Polyangium luteum, an unusual double shoulder appeared. Repair inhibitors like acriflavine, caffeine, and coumarin reduced the survival of UV-irradiated cells if the drugs were incorporated in the post-irradiation plating medium. The shoulders were reduced, but the final inactivation slopes were not affected by the repair inhibitors. Those strains that were resistant to UV were also more resistant to being killed by nitrosoguanidine. A variety of drug-resistant mutants occurred. The spontaneous mutation frequencies to drug resistance varied with the drug and the strain used. Drug-resistant mutants were inducible by UV irradiation and nitrosoguanidine. The UV mutability of Myxococcus xanthus was high compared to Cystobacter sp. However, the nitrosoguanidine mutability of M. xanthus was low compared to the other strains.     

Cytoplasmic membrane proteins of spectinomycin-susceptible and -resistant strains of Neisseria gonorrhoeae.

Cytoplasmic membranes were isolated and examined from two spectinomycin-susceptible and three spectinomycin-resistant clinical strains of Neisseria gonorrhoeae. A laboratory-derived spectinomycin-resistant mutant, obtained by serial passage on gradually increasing concentrations of the antibiotic, and a susceptible revertant, spontaneously arising from one of the resistant clinical strains, were also studied. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis revealed that a major protein, comprising about 7% of total cytoplasmic membrane protein (molecular weight 24,000), was absent in the three clinically isolated spectinomycin-resistant strains. In a revertant, this protein reappeared. During treatment of one of the susceptible strains with spectinomycin, the protein disappeared. However, this correlation was not maintained in the laboratory-derived spectinomycin-resistant mutant. This mutant was of comparable resistant to the clinical isolates, but the 24,000-molecular-weight protein was present in normal quantities. In addition, spectinomycin resistant in clinical isolates was variable compared with stable resistance exhibited by the laboratory-derived mutant. These findings suggested that differences in laboratory-derived versus clinical spectinomycin resistance may be due to different types of resistance mutations.     

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.