Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

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Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

<p>Human noroviruses (HuNoVs) are the leading cause of an acute form of non-bacterial gastroenteritis, where strains belonging to genogroup (G) II are dominant. Upon expression with the baculovirus culture system, virus-like particles (VLPs) of HuNoVs are expected to assemble into T = 3 icosahedral capsid particles resembling the structure of the infectious virion particles. However, some strains were found to assemble into either T = 1 or T = 4 capsids, or a combination of two different capsid forms. In this study, we showed that VLPs of the Virginia 1997 387 (VA387) GII.4 outbreak strain assembled into T = 1, T = 3, and T = 4 capsids upon expression in insect cell culture, the first case for a naturally occurring HuNoV strain to assemble into all three capsid states. TEM analysis revealed that T = 1 icosahedral particles were the most abundant in purified samples, which contrasts previous findings where either T = 3 or T = 4 were the most abundant. We resolved the cryo-EM structures of the T = 1 shell (S) domain, T = 3, and T = 4 particles to 2.24, 2.44, and 3.43 Å, respectively, making them the most resolved norovirus (NoV) structures to date. Single particle cryo-EM 3D analysis showed that the protruding (P) domain of T = 1 and T = 4 VLPs are highly dynamic. Additionally, we showed that T = 3 VLPs are resistant while T = 1 and T = 4 VLPs are sensitive to digestion in the presence of trypsin. This suggested that T = 1 and T = 4 capsids are less stable among the VLPs, which is consistent with the highly dynamic P domain inferred from our cryo-EM 3D analysis. During infection, HuNoVs travel through the gastrointestinal (GI) tract where they encounter a broad range of variable conditions that include pH, ionic strength, and host defenses (e.g., proteases). Our analyses suggest that virions are T = 3 particles as they can survive the GI tract upon exiting the host. We determined the first cryo-EM structure of T = 3 VLPs in complex with the known HuNoV host cell receptor, histo-blood group antigen (HBGA), to a resolution of 2.51 Å, demonstrating that NoV VLPs can serve as a platform in the structural characterization of small ligand molecules. Lastly, we identified a histidine residue retained in the S domain of all identified caliciviruses critical in the assembly of capsids. Our structures and their characterization will contribute to the development of therapeutic agents to combat noroviruses. </p>

Viruses are entities which are made of a few genes and are reliant on obligate parasitism to propagate. Due to the obligate connection to their hosts, virus evolution is constrained to the type of host. Viruses however do transmit to evolutionary distinct hosts; in these cases, the phylogenetic relationship of the hosts usually are close. In some instances, RNA-viruses have made host jumps between evolutionary distant hosts, such as the host jump from invertebrates to vertebrates, and fungi to arthropod. Partitiviruses are double stranded RNA viruses which mainly infect fungi and plants. The defining characteristic of these double stranded RNA viruses are the double layered capsids which are formed by a single open reading frame (ORF). The capsid proteins form icosahedral virus particles which are in the magnitude of 30-40 nm. Metagenomic studies have discovered partitiviruses originating from an insect in the Odanata family, a finding which contradicts the fungal host specificity of partitiviruses. The finding of the Hubei.PLV 11 thus implies the existence of a partitiviruses containing structural elements in their capsids which could be involved in the infection of arthropods. Thus, this virus could be used as a model for a structural comparison with its fungi infecting relatives with hopes to identify common viral structural factors necessary for the infection of arthropods. For this purpose, the Hubei.PLV ORF was cloned and then transfected into insect Spodoptera frugiperda (Sf-9) cells using a baculovirus expression system, “bac-to-bac” expression system. The FLAG-tagged capsid proteins were expressed by the Sf-9 cells to be approximately 60 kDa. After ultra-centrifugation in a sucrose gradient, some spontaneous assembly into the expected ~40 nm icosahedral virus-like particles were observed using low resolution scanning electron microscopy. The observed particles were also confirmed by a dynamic light scattering experiment (DLS) and a higher resolution cryo-EM microscope. Thus, the bac-to-bac expression system can be used to produce VLPs from this genus of viruses, and this metagenomically derived virus genome. However, for future success in defining a high-resolution model of this virus, it is recommended that the Sf-9 culture volume is sufficiently high for enough particle production which is necessary for a high-resolution map. The other virus, the Southern pygmy squid Flavilike virus (SpSFV) has been suggested to be the oldest relative of the land based flaviviruses. The SpSFV was found to be the most divergent of the flaviviruses, and to infect invertebrates. Solving for the structure of the SpSFV and comparing it to vertebrate infecting flaviviruses could therefore lead to the identification of factors necessary for the adaptation to vertebrates and thus the humoral immunity by flaviviruses. The soluble E-protein was expressed using the bac-to-bac expression system. The protein was indicated to be multiglycosylated and approximately 50 kDa which is in line with other strains in the genus. Affinity chromatography did not elute this protein, likely due to the His-tag not being spatially available. Cation exchange could elute some protein, but not much from the small ~30 mL culture. To conclude, VLP assembly was confirmed by the Hubei.PLV, thus, solving for the structure is a distinct possibility when a larger Sf-9 culture is used to produce the VLPs. For the SpSFV soluble E-protein, the protein is secreted into the supernatant of the Sf-9 cultures, making purification a possibility. For this, a large Sf-9 culture can be used to produce this protein and then purify it with a cat-ion exchange chromatography.

Chez la guêpe parasitoïde Venturia canescens, des particules virales dépourvues d'ADN appelées VLP (pour "Virus-like Particules") sont produites spécifiquement dans les ovaires et tapissent le chorion des oeufs qui sont injectés dans la chenille hôte. Les VLP ont une fonction immunosuppressive pour l'hôte parasité et permettent ainsi la survie des oeufs du parasitoïde. Ces VLP résultent de l’intégration d’un nudivirus dans le génome de l’ancêtre de la guêpe, nudivirus qui a été ensuite domestiqué pour former des liposomes viraux capables de véhiculer dans l’hôte des protéines de virulence d'origine cellulaire. L’étude réalisée au cours de cette thèse a eu pour objet, d’une part, d'étudier les mécanismes de domestication virale qui ont conduit au virus symbiotique endogène actuel nommé VcENV (pour V. canescens endogenous nudivirus) et d’autre part, d'apporter des éléments de réponse sur le processus de morphogénèse et le mode d'action parasitaire des VLP. / Viral particles devoid of DNA called VLPs (for Virus-Like Particles) are specifically produced in the ovaries of the parasitoid wasp Venturia canescens and line the chorion of the wasp’s eggs injected into the host caterpillar. VLPs are immunosuppressive and allow parasitoid eggs survival. These VLPs result from the integration of a nudivirus into the wasp ancestor genome, nudivirus which was then domesticated to form viral liposomes capable of carrying, into the host, virulence proteins of cellular origin. The aim of the study carried out during this thesis was, first, to analyze the viral domestication mechanisms that led to the current endogenous symbiotic virus called VcENV (for V. canescens endogenous nudivirus) and secondly to provide some answers on VLPs morphogenesis process and parasitic mode of action.