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Resistance of human viral pathogens to various antiviral drugs is a serious medical problem. Two modes of drug resistance in cytomegalovirus infections have been observed, the first being altered (decreased) drug metabolism by the infected cells, and the second reduced sensitivity of the viral deoxyribonucleic acid polymerase enzyme to the active form of the drug. Mice infected with the murine cytomegalovirus have been used extensively as an animal model for the human cytomegalovirus, and drug-resistant strains in this model have been identified. To better understand the mode of drug resistance of the virus, the metabolism of two antiviral drugs, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (ganciclovir) and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (cidofovir), was studied in cells infected with the virus. The degree of resistance of the mutant virus strain to these two drugs and also to the drug phosphonoformic acid (foscarnet) was measured in viral plaque reduction assays. The resistant strain was 14-,4-, and 11-fold less sensitive to the drugs ganciclovir, foscarnet, and cidofovir, respectively, than a sensitive (wildtype) strain Metabolism of the antiviral drugs ganciclovir and cidofovir was studied in C127I mouse mammary tumor cells infected with the mutant strain. Uninfected C127I cells and C127I cells infected with the sensitive strain of murine cytomegalovirus were used as controls. The cells were treated with tritium-labeled ganciclovir or cidofovir and studied under a variety of parameters. Among these were duration of treatment, multiplicity of infection, and concentration of compound. After incubating, the cells were acid extracted and analyzed with high-pressure liquid chromatography. The radioactivity of each sample was measured on a scintillation counter and converted into picomoles of drug per million cells. No significant difference was observed between the virus strains in terms of metabolism or catabolism of the two drugs. This effect remained constant, even when controlling for parameters such as the amount of virus infecting each cell, duration of treatment, or concentration of drug. Based on these results, it appears that the mode of resistance in this mutant strain of virus to ganciclovir and cidofovir is not due to an alteration in metabolism of these two compounds by infected cells. Thus, it is proposed that drug resistance in this mutant strain of virus is due to altered viral deoxyribonucleic acid polymerase function.

The emergence of viruses with zoonotic potential, i.e. with the potential ability to cross species barriers to infect unnatural hosts, poses a huge threat to humans. It is therefore essential to develop new methodologies to rapidly and efficiently generate attenuated virus vaccine candidates to attempt to control the threat. Viruses need to be able to at least partially inhibit the host’s innate defence mechanism, known as the interferon (IFN) system, to replicate efficiently in vivo and establish a productive infection. It has been previously reported that viruses that have lost their ability to circumvent the host’s IFN response, or IFN-sensitive viruses, are promising candidates for live attenuated virus vaccines. Here we report on the development of a cell-based method to attempt to rapidly select IFN-sensitive viruses that can not block IFN signalling, from wild-type virus populations. Lentivirus vectors containing selection markers (HSV-tk – Herpes Simplex virus thymidine kinase gene and pac – puromycin resistance gene) under the control of a tight IFN-inducible promoter (the murine Mx1 promoter) were generated and used to specifically engineer HEp2 cell lines, termed Mx GIPSE and Mx TIPSE, for this purpose. The developed methodology relies on the engineered cell lines and a selection procedure using exogenous IFN-α and puromycin: if a cell is infected with IFN-resistant virus, it will die in the presence of IFN-α and puromycin because IFN signalling will be blocked, thereby blocking the activation of the Mx1 promoter and consequent expression of pac; if a cell is infected with an IFN-sensitive virus, it will survive in the presence of IFN-α and puromycin because the Mx1 promoter will become activated through the IFN signalling pathway, leading to the expression of pac. IFN-sensitive viruses can then be rescued from the surviving cells, and amplified using IFN-permissive cell lines expressing viral IFN antagonist proteins (proteins that block the host’s IFN response). When tested on PIV5 strains CPI- (an IFN-sensitive virus) and CPI+ (an IFN-resistant virus), the developed method allowed the survival and amplification of cells infected with CPI-, whilst cell death was observed for cells infected with CPI+. Whilst the developed methodology seems promising, further developments of the system are required. The possibilities of using the developed methodology in combination with other techniques, such as FACS sorting and immune selection, to rapidly select IFN-sensitive mutant viruses from wild-type and mutagenised virus populations are discussed. The potential to use Mx TIPSE cells to select IFN-resistant revertant viruses from IFN-sensitive virus populations is also discussed. In addition, a high throughput screening assay has been developed using the engineered Mx GIPSE and Mx TIPSE cell lines to search for compounds that block IFN signalling or that block the action of viral IFN antagonist proteins. Compounds that block IFN signalling would potentially be useful as anti-inflammatory drugs whilst compounds that block the action of viral IFN antagonist proteins would be valuable as antiviral drugs.

The outbreak of pandemic influenza and its ability to spread rapidly makes it a severe threat to public health. Antiviral drugs such as oseltamivir (Roche’s Tamiflu™) and zanamivir (GlaxoSmithKline’s Relenza™) are neuraminidase (NA) inhibitors (NI), which bind more tightly to NA than its natural substrate, sialic acid. However, the virus can acquire resistance to antiviral drugs by developing single point mutations (such as H274Y) in the target protein. Thus in some cases the drugs may not be as effective as expected. The high level of inconsistency exhibited by fluorometric assays and the short half-life of the chemiluminescent assay for monitoring drug resistance lead to the need for a simple, label-free, reliable assay. To address this problem, this work focused on three main objectives: 1) to determine the binding affinities of two common anti-viral drugs (oseltamivir and zanamivir) against the influenza NA wild type and drug resistant mutants using bioinformatics software Schrodinger Suite™ 2010. 2) To develop a reliable label-free, real-time, surface plasmon resonance (SPR) assay to measure the binding affinity between influenza viral coat protein neuraminidase (wild type and mutant) and anti-viral drugs. 3) To develop an SPR inhibition assay to quantitatively compare the interactions of sialic acid, zanamivir and oseltamivir with the viral coat protein neuraminidase (wild type and mutant). The entire docking process was carried out using Schrödinger Suite™ 2010. The 2009 pandemic H1N1 neuraminidase (PDB: 3NSS) was used throughout the docking studies as the wild type structure. Five mutants (H274Y, N294S, H274N, A346N and I222V) and three ligands (sialic acid, oseltamivir and zanamivir) were built using the maestro module. The grid-based ligand docking with energetics (GLIDE) module and induced fit docking (IFD) module were used for docking studies. The binding affinities, Gibbs free energy change (∆G) and molecular mechanics-generalized born energy/ solvent accessible area (MM-GB/SA) values for wild-type NA interactions show that both the antiviral drugs studied interact strongly with the wild-type protein. The ∆G values for all antiviral interactions with mutant NA forms were reduced in magnitude, thereby indicating that they are less favourable than interactions with the wild-type protein. A similar trend was observed with MM-GB/SA results. Amongst all of the computed values, MM-GB/SA was the closest to the experimental data. In several cases, the interactions between the anti-viral drugs and NA mutants were markedly less favourable than those between sialic acid and the same mutants, indicating that these mutations could confer anti-viral resistance. Influenza NA wild-type and H274Y mutant were expressed in baculovirus expression system (BVES) in insect cells. The expressed proteins were partially purified using the standard purification techniques of anion exchange and size exclusion chromatography (SEC). A fluorometric activity assay was performed on the recombinant proteins. Both the wild type and the mutant showed similar level of activities. In addition, the recombinant NAs were used in an inhibition assay. Oseltamivir was found to be sensitive to wild type protein (IC50 = 0.59 nM) and resistant to the H274Y mutant protein (IC50 = 349.43 nM). On the other hand, zanamivir was sensitive to both wild type (IC50 = 0.26 nM) and the H274Y mutant (IC50 = 0.44 nM). This indicated that zanamivir was a more potent inhibitor than oseltamivir. These findings were in good agreement with the literature. An SPR assay for accurate monitoring of influenza antiviral drug resistance was developed. A spacer molecule (1, 6- hexanediamine) was site-specifically tethered to the inert 7-hydroxyl group of zanamivir. The tethered zanamivir was immobilized onto an SPR GLC chip to obtain a final immobilization response of 431 response units (RU). The reference subtracted binding responses obtained for NA wild-type and H274Y mutant were analysed using the ProteOn Manager™ Software tools. The SPR curves were fitted to a simple Langmuir 1:1 model with drift to obtain association rate constant (ka) and dissociation rate constants (kd). The relative binding values obtained from literature and the current SPR assay (1.9 and 1.7 respectively) suggested that the current SPR assay yielded similar results to the existing labelled enzymatic assay. In addition, an SPR inhibition assay was developed. The calculated IC50-spr values were compared and it was observed that oseltamivir was sensitive to wild type protein (IC50-spr = 7.7 nM) and resistant to the H274Y mutant protein (IC50-spr = 256 nM). On the other hand, zanamivir was sensitive to both wild type (IC50-spr = 2.16 nM) and the H274Y mutant (IC50-spr = 2.4 nM). Sialic acid was also found to be sensitive to both wild type (IC50-spr = 5.5 nM) and H274Y mutant (IC50-spr = 3.25 nM). In the cases studied, the viral proteins remained sensitive to sialic acid, consistent with retention of virulence of these mutant strains. It was concluded that zanamivir is a more potent inhibitor than oseltamivir for treating the H274Y mutant. Comparison of the SPR inhibition results with the docking results revealed a similar trend. The wild-type NA and H27Y mutant retained binding affinity for sialic acid and zanamivir. Oseltamivir showed a significant decrease in binding affinity for the H274Y mutant compared with the wild-type. This was because of the disruption of the salt bridge formation within NA that was vital for oseltamivir activity. To my knowledge, this is the first SPR biosensor assay developed to monitor influenza antiviral drug resistance. There is a tremendous scope to extend this study to more mutants and new antiviral drugs. This could pave the way for a reliable SPR biosensor assay to replace low consistency labelled enzymatic assays.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and toxikology Student: Zuzana Matiašková Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Role of selected ABC and SLC transporters in transmembrane permeability of maraviroc: effect on transport in placenta Antiretroviral drug maraviroc is an inhibitor of CCR5-trophic HIV virus and belongs to the group of entry inhibitors. Nowadays, maraviroc is administered as part of combination antiretroviral therapy (cART) primarily in adults, children over the age of two and pregnant women to reduce the risk of transmission of HIV to the fetus. The knowledge of interactions of maraviroc with drug transporters in placenta is crucial for optimizing the therapy during pregnancy, both in terms of efficacy and potential adverse effects. Maraviroc is known substrate of ABCB1 transporter, which plays a protective role to the fetus by its efflux activity in the apical membrane of trophoblast. However, the results of recent study employing dually perfused human placenta suggest involvement of other transport mechanisms in the maraviroc transplacental pharmaocokinetics, especially those operating in the opposite direction to ABCB1. The aim of this study was to evaluate in vitro studies whether, besides ABCB1,...

Mouse monoclonal antibodies directed against Pichinde virus (PCV) were produced to evaluate their application as vehicles for the delivery of antiviral drugs or toxins to virus-infected cells. Four monoclonal antibodies, PC4.9A6, PC4.9D3, PC4.7C2, and PC4.8D3, were of the IgG2a subisotype and reacted with acetone-fixed and live PCV-infected Vero-76 cells. In vivo stained splenic macrophages derived from PCV-infected hamsters that had been injected with fluorescein-labeled PC4.9A6 (FITC9A6) demonstrated a 400% increase in total fluorescence over similarly treated, non-infected cells when analyzed by flow cytometry. This is an indication that FITC-9A6 does have some ability to specifically target PCV infected cells in vivo. Radioimmunoprecipitation of viral proteins showed that all the antibodies precipitated two different PCV proteins, one of 64,000 daltons and another of 38,000 daltons. These proteins are, respectively, PCV ix nucleoprotein (NP) and a breakdown product of NP that is present in PCV infected cells. An immunofluorescent assay (IFA) for PCV was developed. This IFA was used for antiviral drug assays against PCV. The assay was performed by adding fluorescein-labeled anti-PCV monoclonal antibody to fixed, virus infected cells at 24 h after infection and counting the fluorescent cells. The 50% effective dose (EDso) for ribavirin against PCV using this IFA was 6.0 IJ. g/ml. The EDso of ribavirin using inhibition of marginal PCV cytopathogenic effect after 12 days was 6.0 IJ. g/ml and using plaque reduction after 5 days is 2.5 IJ. g/ml, indicating that this IFA was of comparable sensitivity. An immunotoxin (IT) was produced by the conjugation of gelonin to PC4.9A6. This IT was tested in vivo in PCV-infected MHA hamsters. It was not active against the disease at the dosage tested and by the intraperitoneal (i.p.) treatment route employed in this study. The positive control, ribavirin, administered i.p. for 14 days at a dosage of 40 mg/ ml significantly increased the number of survivors. Three of 5 IT toxicity control animals developed some humoral response that inhibited PC4.9A6 binding to infected cells. They did not show any humoral response to the gelonin moiety of the IT.

The concern for pharmaceutically active compounds (PhACs) as contaminants in the environment and the need to assess their environmental risk have greatly increased since the early nineties. Among PhACs, antibiotics and antiviral drugs are of important concern due to their role in growing antibiotic and antiviral drugs resistance among pathogenic bacteria and influenza viruses, respectively. Besides resistance issue, the compounds may upset sensitive ecosystems as they are designed to be highly bioactive. Clinically-important antibiotics are virtually ubiquitous contaminants in sewage water and surface water. Notably, recent emergence of novel influenza and use of anti-influenza drugs (specially Tamiflu®) during seasonal influenza、 influenza epidemics and for future pandemic are of emerging concern. Every year seasonal influenza epidemic causes tens of millions of respiratory illnesses and 250, 000 to 500, 000 deaths worldwide. WHO (World Health Organization) recommend the use of antiviral drug Tamiflu® during pandemic, as they are easy to use. Currently only Japan uses over eighty percent of Tamiflu® prescribed globally during common seasonal influenza. It is a fact that a huge amount of antiviral drugs and antibiotics ( for post infection cure of respiratory illness) will be used during an influenza pandemic and will arrive to sewage treatment plants (STPs).Unfortunately, these compounds behaviors are mostly unknown in both conventional and advanced STPs. The exposure of antiviral drug in the wild fowl gut and its implications for hastening the generation of antiviral-resistance in avian influenza viruses are also an emerging issue. The major objective of this thesis work was to investigate the occurrence of antibiotics and antiviral drugs in sewage treatment plants and their fate in different sewage treatment plants. The specific objectives were as follows: (a) to established appropriate analytical method for the selected antibiotics and antiviral drugs in sewage treatment plants, (b) to investigate the occurrence and removal of antibiotics and antiviral drugs in sewage treatment plants differ in technology and operation conditions; and (c) to predicts environmental concentration of the target compounds during a pandemics and appraisal of appropriate technology to reduce the risk associated with widespread use under pandemic conditions. In this study we selected twenty antibiotics: one beta-lactam: ampicillin; four macrolides: azithromycin, clarithromycin and roxithromycin; five quinolones: ciprofloxacin, enrofloxacin, levofloxacin, nalidixic acid and norfloxacin; two tetracycline: tetracycline and oxytetracycline; five sulfonamides: sulfadimethoxine, sulfadimizine, sulfamerazine, sulfam- ethoxazole and sulfamonomethoxine; and four others: lincomycin, novobiocin, salinomycin and trimethoprim. Oseltamivir Carboxylate (OC), the active metabolite of oseltamivir phosphate (Tamiflu®) and amantadine (AMN) were selected as antiviral drugs. This dissertation consists of nine chapters: Chapter I describe the background and objective of the study and chapter II represent a brief literature review. In Chapter III, analytical methods for selected antibiotics and antiviral drugs (for the first time) in water and wastewater were described. In Chapter IV, the occurrences and fate of antibiotics in sewage treatment plants were investigated in Japan and China. Clarithromycin was detected in the highest concentration in influent (1129 to 4820　ng/L), followed by azithromycin (160 to 1347　ng/L), levofloxacin (255 to 587　ng/L) and norfloxacin ( 155 to 486 ng/L) and sulfamethoxazole (159 to 176ng/L) in Japan. Ozonation as tertiary treatment of secondary effluent for wastewater reclamation provided significant elimination of antibiotics. Fifty present of the selected antibiotics were removed over eighty percent during ozonation. There was no elimination of antibiotics in dissolve phase during ultra filtration. From Chapter IV a hypothesis was drawn on antibiotics removal and its relation with longer sludge retention time (SRT) in STPs and in Chapter V the role of nitrifier in antibiotics removal was evaluated to verify the hypothesis established from Chapter IV. Nitrifying activated sludge (NAS) can biodegrade the tested antibiotics with different biodegradation rate between　2.74 to 9.95 L/gSS/d. Sulfamethoxazole and sulfamerazine degraded faster than trimethoprim, clarithromycin and enrofloxacin. In Chapter VI, occurrence of antiviral drugs in sewage water discharge and in river water in Japan was conducted during seasonal influenza epidemic and their fate in different sewage treatment facilities were evaluated in Chapter VII. This is the fist findings of antiviral (anti-influenza) drugs in the environment in the world and for the first time the removal mechanism in STPs was elucidated. Finally, it was observed that only primary and secondary treatment processes in STPs were not sufficient to remove these compounds significantly. Overall OC and AMN removal in STP with ozonation as tertiary treatment was 90% and 96% respectively. In ozonation batch experiment, Chapter VIII, ( feed ozone gas concentration 4.0mg/L, ozone gas flow rate 0.23L/min to maintain ozone feed rate of 0.6 mg/L/min), it was observed that AMN and OC concentration decreased linearly with time in all the experiments conducted and it can be, therefore, said that the degradation reactions follow pseudo first-order reaction. The k'O3 (pseudo first-order rate constant for O3) of AMN was 0.596/min (0.00993/sec), and OC was 0.524 /min (0.008725/sec) and over 99% removal within 10min. Chapter VIII described the predicted OC and antibiotics concentration in STPs influent, secondary effluent, after advance tertiary treatment (ozonation) and receiving water during a pandemic with three expected infection scenario ( according to US CDC FluAid model 2.0) in Kyoto city. Both antiviral drugs and antibiotics pose an environmental risk associated to there widespread use during a future pandemic. Ozonation as tertiary treatment can provide a technological solution to reduce the ecotoxicological effect of antibiotics and antiviral drugs uses during a pandemic. In a full scale STP, the antiviral drugs (OC and AMN) reduction were over 90% from secondary effluent after ozonation during seasonal influenza outbreak in Kyoto city in 2008/2009. Finally, (1) analytical methods for commonly used antibiotics and antiviral drugs in water sample was developed with an excellent precision and accuracies, (2) both antibiotics and antiviral drugs were detected in environmental sample, and their behavior in STPs were elucidated. Antivirals in this study were the first time findings in sewage water. This study will provide a surrogate for planning a pandemic preparedness action plan for sewage treatment pants for ecotoxicological risk management. / Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第14931号 / 工博第3158号 / 新制||工||1474(附属図書館) / 27369 / UT51-2009-M845 / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 伊藤 禎彦, 教授 藤井 滋穂 / 学位規則第4条第1項該当

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and toxikology Student: Zuzana Matiašková Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Role of selected ABC and SLC transporters in transmembrane permeability of maraviroc: effect on transport in placenta Antiretroviral drug maraviroc is an inhibitor of CCR5-trophic HIV virus and belongs to the group of entry inhibitors. Nowadays, maraviroc is administered as part of combination antiretroviral therapy (cART) primarily in adults, children over the age of two and pregnant women to reduce the risk of transmission of HIV to the fetus. The knowledge of interactions of maraviroc with drug transporters in placenta is crucial for optimizing the therapy during pregnancy, both in terms of efficacy and potential adverse effects. Maraviroc is known substrate of ABCB1 transporter, which plays a protective role to the fetus by its efflux activity in the apical membrane of trophoblast. However, the results of recent study employing dually perfused human placenta suggest involvement of other transport mechanisms in the maraviroc transplacental pharmaocokinetics, especially those operating in the opposite direction to ABCB1. The aim of this study was to evaluate in vitro studies whether, besides ABCB1,...

Influenza virus A and B are common pathogens that cause respiratory disease in humans. Recently, a highly virulent H5N1 subtype avian influenza virus caused disease outbreaks in poultry around the world. Drug resistant type A viruses rapidly emerged, and the recent H5N1 viruses were reported to be resistant to all current antiviral drugs. There is an urgent need for the development of new antiviral drugs target against both influenza A and B viruses. This dissertation describes work to identify small molecule inhibitors of influenza protein NS1 by a high throughput fluorescence polarization assay. The N-terminal GST fusion of NS1A (residue 1-215) and NS1B (residue 1-145) were chosen to be the NS1A and NS1B targets respectively for HT screening. In developing the assay, the concentrations of fluorophore and protein, and chemical additives were optimized. A total of 17,969 single chemicals from four compound libraries were screened using the optimized assay. Six true hits with dose-response activity were identified. Four of them show an IC₅₀ less than 1 [micromolar]. In addition, one compound, EGCG, has proven to reduce influenza virus replication in a cell based assay, presumably by interacting with the RNA binding domain of NS1. High throughput, computer based, virtual screenings were also performed using four docking programs. In terms of enrichment rate, ICM was the best program for virtual screening inhibitors against NS1-RBD. The compound ZINC0096886 was identified as an inhibitor showing an IC₅₀ around 19 [micromolars] against NS1A, and 13.8 [micromolars] against NS1B. In addition, the crystallographic structures of the NS1A effector domain (wild type, W187A, and W187Y mutants) of influenza A/Udorn/72 virus are presented. A hypothetical model of the intact NS1 dimer is also presented. Unlike the wild type dimer, the W187Y mutant behaved as a monomer in solution, but still was able to binding its target protein, CPSF30, with wild type binding affinity. This mutant may be a better target for the development of new antiviral drugs, as the CPSF30 binding pocket is more accessible to potential inhibitors. The structural information of those proteins would be very helpful for virtual screening and rational lead optimization. / text