Thesis (Doctoral)--Justus Liebig-Universität Giessen, 1982.

No description available.

No description available.

Neuraminidase (NA) inhibitors are one of two classes of antiviral compounds available for the control of influenza infections. The H275Y NA mutation confers resistance to the NA inhibitor oseltamivir carboxylate among the N1 influenza subtype and has been identified from resistant variants with distinct epidemiological outcomes in human. Specifically, dominance of oseltamivir-resistant variant of the A/Brisbane/59/2007-like viruses was reported during the 2008 to 2009 influenza season, until it was replaced by the 2009 pandemic H1N1 virus [A(H1N1)pdm09]. Since the emergence of the 2009 pandemic, the fitness and transmission potential of oseltamivir-resistant A(H1N1)pdm09 variants carrying the H275Y mutation has been a concern. This project aims to systematically evaluate the fitness of viruses carrying the H275Y mutation for A(H1N1)pdm09 and seasonal H1N1 viruses. A panel of recombinant viruses with their NA gene derived from the A(H1N1)pdm09 A/California/04/09 (CA04), seasonal H1N1 A/New Caledonia/20/1999 (NewCal) or A/Brisbane/59/2007 (Brisbane) was generated in the genetic background of CA04. The H275Y mutation in all three viruses led to a reduced affinity for 3’-sialylactose (3’SL) or 6’-sialylactose (6’SL). Similarly, lowered enzyme activity was observed across H275Y-carrying viruses in 3’SL and 6’SL, with the exception of RG-CA04NA-H275Y at catalyzing 3’SL. Differential 3’SL and 6’SL substrate usage was observed between the NA of seasonal H1N1 and A(H1N1)pdm09 viruses. Reduced infectivity was also observed for recombinant CA04 viruses carrying the H275Y mutation with decreased infectivity in mucin-secreting primary human airway epithelial cells when compared to their oseltamivir-sensitive counterparts. In the ferret model, the pathogenicity of RG-CA04NA-H275Y and RG-CA04BrisbaneNA-H275Y viruses was attenuated albeit the transmissibility was minimally affected when compared to RG-CA04 wild-type virus. In parallel, recombinant seasonal H1N1 viruses encoding the surface glycoproteins of NewCal and Brisbane were tested in ferrets. Results indicated that NewCal and Brisbane viruses carrying the H275Y mutation displayed comparable transmission efficiencies to the wild-type NewCal virus via direct-contact and respiratory-droplet settings. These results suggest that the H275Y NA mutation only leads to a minor reduction in viral fitness, with its transmission potential being minimally affected in the naïve ferret model. / published_or_final_version / Public Health / Master / Master of Philosophy

Les infections à virus influenza demeurent un problème de santé majeur dans le monde entier. Une nouvelle classe d'agent anti-influenza servant au traitement et à la prévention a été développée, soit les inhibiteurs de neuraminidase. L'objectif premier de ce projet de maîtrise était de générer différents variants du virus influenza A/WSN/33 (HlNl) résistants au peramivir et au zanamivir. En second lieu, nous avons étudié l'effet de certaines mutations du gène NA, en utilisant des protéines NA recombinantes, quant à leur degré de susceptibilité face à différents inhibiteurs de neuraminidase. La virulence d'un virus A/WSN/33 généré par génétique inverse contenant les mutations H274Y et N294S dans le gène NA, a été étudiée dans un modèle murin Balb/c. Nous avons également caractérisé des virus A/H3N2 isolés d'un enfant immunosupprimé traité avec trois antiviraux (oseltamivir, amantadine et zanamivir). / Influenza infections remain a major health problem Worldwide. For the treatment and prevention of influenza infections, a new class of anti-influenza agents, the neuraminidase inhibitors (NAIs), has been developed. The objectives of this master project were firstly the in vitro generation of peramivir and zanamivir-resistant variants. Secondly, we have studied the effect of selected NA mutations on the susceptibility profiles to different NAIs using recombinant NA proteins. We have also investigated the virulence of reverse genetics-rescued A/WSN/33 viruses harboring H274Y and N294S NA mutations in a Balb/c model. Finally, we studied influenza A/H3N2 viruses isolated from an immunocompromised child who was treated with three antivirals (oseltamivir, amantadine and zanamivir).

No description available.

No description available.

Two influenza viruses, the virulent clone 7a (H3N2) and the attenuated A/Fiji/15899/83 (H1NI) (A/Fiji), induce different level ofapoptosis in both MDCK and HeLa cells. Apoptosis induced by these two viruses could be partially inhibited by two NA inhibitors (GGI67 and DANA), when present during virus entry, but not subsequently. GGI67, which cannot enter cells, did not affect the level of infection. These results suggest that NA plays a role in influenza virus-induced apoptosis and that it acts early in the replication cycle. However, ammonium chloride, which prevents virus entry reduced the level of apoptosis induced by both viruses. In addition, amantadine, which prevents virus uncoating, inhibited apoptosis induced by the amantadine-sensitive strain A/Udorn/307/72 (H3N2). Therefore, other viral components acting intracellularly may also be involved in influenza virus-induced apoptosis. Clone 7a, A/Fiji or A/WSN/33 (H1N1)(A/WSN) proteins were expressed in HeLa cells, fused to the Herpes simplex tegument coat protein VP22. This protein has the ability to translocate from the cell in which it is synthesised to surrounding cells. Therefore, VP22- fusion proteins are delivered to a high number of cells, regardless of the level of transfection. A/WSN PB1, PB2 and HA induced significant levels ofapoptosis as did the NA, M1, M2 and NS1 proteins from clone 7a and the M1, M2, NSI and NS2 proteins from A/Fiji. Conversely, clone 7a and A/Fiji NP and A/Fiji PB2 and NA did not induce apoptosis. Confocal microscopy revealed that apoptosis was associated with fusion protein expression and that the location of the proteins corresponded with that expected during influenza virus infection. In addition, the mechanism of clone 7a NA-induced apoptosis was investigated. GGI67 inhibited apoptosis induced by the expression of clone 7a NA. Hence, NA activity is required for the induction of apoptosis. In addition, apoptosis was completely abrogated in the presence of an anti-transforming growth factor (TGF)-B antibody suggesting the activation of TGF-B during the translocation of the fusion protein from one cell to another is involved in clone 7a NA-induced apoptosis. Furthermore, clone 7a and A/Fiji NS1 proteins induced apoptosis when expressed alone, but they inhibited double-stranded (ds) RNA-induced apoptosis. However, the level of apoptosis observed in the presence of dsRNA was not reduced to background levels suggesting a pro and anti-apoptotic action of NS1 in influenza virus-infected cells. The potential mechanisms involved in the induction of apoptosis by each protein and the relevance to infection is discussed.

Thesis (Ph.D.)--University of Western Australia, 2004.

Building upon existing synthetic methods, we have optimized the synthesis of Thiele’s methyl ester to an efficient and scalable methodology. As part of a study of chemo- and regioselective transformations within the Thiele’s ester scaffold, we designed and synthesized a new suite of molecular scaffolds incorporating a broad range (from 123° to 176°) of cleft angles. In addition to this, we compared two competing conceptual models for their ability to rationalize the selective formation of Thiele’s ester and two minor regioisomers which arise during the formation of the target product. We found that radical stabilization arguments (based on Deslongchamps’ seminal work) outperformed the classic frontier molecular orbital theory model in predicting the regioselectivity of Thiele’s ester dimerization. When this method was combined with simple steric arguments, we arrived at a general algorithm to rationalize Thiele type dimerization, including all the known homo- and heterodimerizations in the literature as well as a novel phosphine oxide-containing Thiele acid analogue discovered as part of this thesis work. In order to stimulate the use of Thiele’s ester chemistry in a diverse range of applications, we took advantage of our Thiele’s ester methodology to achieve a mono ester-substituted dicyclopentadiene (colloquially referred to as a “half” Thiele’s ester), and used this as the precursor of a novel functionalized polydicyclopentadiene (fPDCPD) ROMP polymer. The resulting fPDCPD has the highest glass-transition temperature reported for any polydicyclopentadiene material and allows for the facile manipulation of the surface chemistry through alteration of the embedded functional group. A long-term goal in the Wulff lab is to use Thiele’s ester as a scaffold for the generation of conformationally restricted (“peramivir-like”) neuraminidase inhibitors. Setting the groundwork for this, we explored the selectivity of various peramivir derivatives toward group-1 vs. group-2 neuraminidase enzymes. To this end, we coupled a wide range of alkyl chains and aromatic rings with different length and size parameters onto the primary amine of peramivir. We found that our de-guanidinylated peramivir analogues showed a rare target selectivity against group-2 neuraminidases instead of group-1 neuraminidases, which might due to the ring geometry of peramivir as well as the reduced electrostatic interaction between the amino group from our analogues and the Asp147-His150 residues from the enzyme. This suggested that it is possible for group-2 neuraminidases to have a more open 150-cavity state than group-1 neuraminidases. Additionally, the respectable IC50 values for these compounds, together with their significantly reduced polarity (relative to peramivir itself) may prove advantageous from a bioavailability standpoint. / Graduate / 2019-04-30