Molecular and biological characterisation of orthobunyaviruses

Orthobunyaviruses are the largest genus within the Bunyaviridae family, with over 170 named viruses classified into 18 serogroups (Elliott and Blakqori, 2001; Plyusnin et al., 2012). Orthobunyaviruses are transmitted by arthropods and have a tripartite negative sense RNA genome, which encodes 4 structural proteins and 2 non-structural proteins. The non-structural protein NSs is the primary virulence factor of orthobunyaviruses and potent antagonist of the type I interferon (IFN) response. However, sequencing studies have identified pathogenic viruses that lack the NSs protein (Mohamed et al., 2009; Gauci et al., 2010). The work presented in this thesis describes the molecular and biological characterisation of divergent orthobunyaviruses. Data on plaque morphology, growth kinetics, protein profiles, sensitivity to IFN and activation of the type I IFN system are presented for viruses in the Anopheles A, Anopheles B, Capim, Gamboa, Guama, Minatitlan, Nyando, Tete and Turlock serogroups. These are complemented with complete genome sequencing and phylogenetic analysis. Low activation of IFN by Tete serogroup viruses, which naturally lack an NSs protein, was also further investigated by the development of a reverse genetics system for Batama virus (BMAV). Recombinant viruses with mutations in the virus nucleocapsid protein amino terminus showed higher activation of type I IFN in vitro and data suggests that low levels of IFN are due to lower activation rather than active antagonism. The anti-orthobunyavirus activity of IFN-stimulated genes IFI44, IFITMs and human and ovine BST2 were also studied, revealing that activity varies not only within the orthobunyavirus genus and virus serogroups but also within virus species. Furthermore, there was evidence of active antagonism of the type I IFN response and ISGs by non-NSs viruses. In summary, the results show that pathogenicity in man and antagonism of the type I IFN response in vitro cannot be predicted by the presence, or absence, of an NSs ORF. They also highlight problems in orthobunyavirus classification with discordance between classical antigen based data and phylogenetic analysis.

Trapping phosphorus (III) into polyoxometalates: synthesis, characterisation and electronic behaviour study

The introduction of electronically-active heteroanions into polyoxometalates (POMs) is one of the emerging topics in this field. The novel clusters have shown unprecedented intramolecular electron-transfer features that can be directly mediated by the incorporated heteroanions. In this thesis, we will focus on the study of phosphite (HPO32-) as new non-traditional heteroanions, discover HPO32- templated nanostructures, investigate their electronic behaviours as well as understand the self-assembly process of HPO32--templated species. The thesis starts with incorporating HPO32- into POM cages. The feasibility of this work was illustrated by the successful trapping of HPO32- into a “Trojan Horse” type {W18O56} nanocage. The reactivity of embedded {HPO3} was fully studied, showing the cluster undergoes a structural rearrangement in solution whereby the {HPO3} moieties dimerise to form a weakly interacting (O3PH···HPO3) moiety. In the crystalline state a temperature-dependent intramolecular redox reaction and structural rearrangement occurs. This rearrangement appears to proceed via an intermediate containing two different templates, a pyramidal {HPO3} and a tetrahedral {PO4} moiety. {HPO3} templated POM cages were then vigorously expanded and led to the isolation of five either fully oxidised or mixed-valence clusters trapped with mono-, di-, or tri- {HPO3}. Interestingly, an intriguing 3D honeycomb-like host-guest structure was also synthesised. The porous framework was self-aggregated by a tri-phopshite anion templated {W21} cluster with a {VO4} templated Wells-Dawson type {W18} acting as a guest species within the hexagonal channels. Based on this work, we further extended the templating anions to two different redox-active heteroanions, and discovered a unique mixed-heteroatom templated system built by pairing redox-active {HPIIIO3} with {TeO3}, {SeO3} or {AsO3}. Two molecular systems were developed, ie. “Trojan Horse” type [W18O56(HPO3)0.8(SeO3)1.2(H2O)2]8- and cross-shaped [H4P4X4W64O224]32-/36-, where X=TeIV, SeIV, AsIII. In the case of {W18(HPO3)0.8(SeO3)1.2}, the compound is found to be a mixture of heteroleptic {W18(HPO3)(SeO3)} and homoleptic {W18(SeO3)2} and {W18(HPO3)2}, identified by single crystal x-ray diffraction, NMR as well as high resolution mass spectrometry. The cluster exhibited similar temperature-dependent electronic features to “Trojan Horse” type {W18(HPO3)2O56}. However, due to the intrinsic reactivity difference between {HPO3} and {SeO3}, the thermal treatment leads to the formation of an unusual species [W18O55(PO4)(SeO3)]5-, in which {HPO3} was fully oxidised to {PO4} within the cage, whereas and lone-pair-containing {SeO3} heteroanions were kept intact inside the shell. This finding is extremely interesting, as it demonstrated that multiple and independent intramolecular electronic performance can be achieved by the coexistence of distinct heteroatoms within a single molecule. On the other hand, the cross-shaped [H4P4X4W64O224]32-/36- were constructed by four {W15(HPO3)(XO3)} building units linked by four {WO6} octahedra. Each building unit traps two different heteroatoms. It is interesting to note that the mixed heteroatom species show self-sorting, with a highly selective positional preference. Smaller ionic sized {HPO3} are self-organised into the uncapped side of {W15} cavity, whereas closed side are occupied by larger heteroatoms, which is surprisingly opposed to steric hindrance. Density functional theory (DFT) calculations are currently underway to have a full understanding of the preference of heteroatom substitutions. This series of clusters is of great interest in terms of achieving single molecule-based heteroatom-dependent multiple levels of electron transfer. It has opened a new way to design and synthesise POMs with higher diversity of electrical states, which may lead to a new type of Q-bits for quantum computing. The third chapter is focused on developing polyoxotungstate building blocks templated by {HPO3}. A series of building blocks, {W15O48(HPO3)2}, {W9O30(HPO3)} {W12O40(HPO3)2} and hexagonal {W6O18(HPO3)} have been obtained. The first four building blocks have been reported with {SeO3} and/or {TeO3} heteroanions. This result demonstrates {HPO3} has a similar reactivity as {SeO3} and {TeO3}, therefore studying the self-assembly of {HPO3}-based building blocks would be helpful to have a general understanding of pyramidal heteroatom-based molecular systems. The hexagonal {W6O18(HPO3)} is observed for the first time in polyoxotungstates, showing some of reactivity difference between {HPO3} and {SeO3} and {TeO3}. Furthermore, inorganic salts and pH values have some directing influence on the formation and transformation of various building blocks, resulting in the discovery of a family of {HPO3}-based clusters with nuclearity ranging from {W29} to {W106}. High resolution mass spectrometry was also carried out to investigate the cluster solution behaviour and also gain information of building block speciation. It is found that some clusters experienced decomposition, which gives rise to potential building blocks accountable for the self-assembly.