The family Bunyaviridae is the largest known taxonomic group of arboviruses. Four of the five genera possess members which are responsible for serious human and livestock disease. The worldwide distribution of these viruses justify studies which will allow understanding of the replication and transcription cycles within permissive cells. The bunyaviruses have been shown to possess a tripartite single strand RNA genome of negative polarity. Replication is confined to the cytoplasm and the virion envelope is acquired when the genome ribonucleoproteins bud into the golgi. Virus release is presumed to be through exocytosis and ultimately cell lysis. The messenger RNA species of all five genera do not possess a poly-A tail of sufficient length to bind to an oligo(dT) cellulose column. This has made separation of viral transcripts from replicating RNAs difficult. In an effort to achieve this separation, infected cell extracts were centrifuged over 20-40% CsCl gradients which permitted replicating RNA structures to band at a density of 1.32 while cellular and viral mRNAs pellet. Recovery of viral transcripts from the CsCl pelleted RNA required synthesis of a cDNA copy of the virus genome to use as a probe. This was done by an unusual method which employs both genome and antigenomic RNA as templates for reverse transcriptase in a first strand synthesis reaction. Recombinant viral clones were then used in a hybrid selection scheme to recover virus mRNA from pelleted material. After recovery, the messages were visualized on acid urea agarose gels pH 3.5, or used to program an in vitro translation reaction. Using these methods, it was established that each genome segment codes for a single messenger RNA which is most likely capped, and that for at least the mid sized segment, proteins with molecular weights which exceed the coding capacity of the genome are translated from the single message.

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Society has become more and more digitalized. Thru time, consumers have built up a resistance for classic marketing strategies such as advertisement in magazines. The resistance for advertisement hasn’t yet become as strong in the digital world. One of many reasons for that is because the digital world is constantly evolving and changing. This change has forced companies to come up with new solutions in order to differentiate them from the competitors. One strategy that has become popular is viral marketing. Viral marketing comes from digital WOM which means that people talk about the company to friends and family. From a consumers’ perspective, viral marketing is still quite new and that is why the authors have chosen to study consumer behaviour regarding sharing advertising videos. In this study, the authors have chosen to collect the primary data with two different methods, a focus group and a survey research. The purpose for the two different methods is to get an overview of the current behaviour but also to get a deeper knowledge of the individuals’ thoughts and the group dynamics that exist. Results of the study shows that consumer behaviour regarding sharing advertising videos depends on; if the video has a capturing story, if the consumer gets affected or if the individual or someone can get benefits from the video. Who is sharing the video, if the video is talked-about or if the individual has been recommended to watch the video are factors that affect if the consumer wants to watch it and where it allows themselves to be exposed to it.

Picornaviruses replicate in association with cytoplasmic membranes of infected cells. Poliovirus 2C and 2BC play an important role in the formation of membranous vesicles, and induce dramatic changes in membrane trafficking. Theiler’s murine encephalomyelitis virus protein 2C was localized in infected cells using an anti-TMEV-2C antibody. Early upon infection, TMEV 2C was localized in the cytoplasm in an ER-like pattern. At later stages, 2C redistributed to a juxtanuclear site, which represents the viral replication site. Co-localization with the Golgi complex could not be observed. TMEV 2C seems to interact in vitro with reticulon 3, a highly conserved ER-associated protein. It was not possible to confirm a previously identified interaction with AKAP10, a protein kinase anchoring protein, presumably reflecting conformational constraints of the interaction. Two mutations in the AKAP10 binding site of TMEV 2C were identified, which inhibit the completion of the infectious cycle of TMEV. The intracellular changes that occur during TMEV infection were observed. Both actin filaments and microtubules may be used at early stages of infection; however both cytoskeleton components accumulate at the periphery of the cell during late stages of infection. A computer- based analysis has demonstrated that TMEV 2C is highly similar to katanin, a microtubule- severing protein, and may play a similar role in the reorganization of microtubules during infection. The Golgi complex turns from a solid, crescent-shaped organelle, into a series of punctuate fluorescent points forming an expanding balloon-like structure surrounding the concomitantly expanding site of virus replication. The remnants of the Golgi complex are finally dispersed throughout the cytoplasm. Live imaging confirmed these findings. It was observed that PKA also undergoes displacement to the cell periphery during infection. However, BIG1 seems to locate to the viral replication site during infection, suggesting it may play a role during viral replication. The localization of PKA and BIG1 in the infected cell may in part explain the observed dispersion of the Golgi complex.

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae. It has a tripartite genome consisting of negative sense RNA segments called large (L), medium (M) and small (S). The S segment encodes the nucleocapsid protein (N) of 233 amino acids. The N protein encapsidates all three segments to form transcriptionally active ribonucleoproteins (RNPs). The aim of this project was to determine the domain map of BUNV N protein. To investigate residues in BUNV N crucial for its functionality, random and site- specific mutagenesis were performed on a cDNA clone encoding the BUNV N protein. In total, 102 single amino acid substitutions were generated in the BUNV N protein sequence. All mutant N proteins were used in a BUNV minigenome system to compare their activity to wt BUNV N. The mutant proteins displayed a wide-range of activity, from parental-like to essentially inactive. The most disruptive mutations were R94A, I118N, W134A, Y141C, L177A, K179I and W193A. Sixty-four clones carrying single substitutions in the BUNV N protein were used in the BUNV rescue system in an attempt to recover viable mutant viruses. Fifty recombinant mutant viruses were rescued and 14 N genes were nonrescuable. The 50 mutant viruses were characterized by: titration, protein labelling, western blotting, temperature sensitivity and host-restriction. Mutant viruses displayed a wide range of titers between 10³ -10⁸ pfu/ml, and three different plaque sizes large, medium and small. Protein labelling and western blotting showed that mutations in the N gene did not affect expression of the other viral genes as much as affecting N protein expression. It was demonstrated that single amino acid substitutions could alter N protein electrophoretic mobility in SDS- PAGE (e.g. P19Q and L53F). Temperature sensitivity tests showed that recombinant viruses N74S, S96S, K228T and G230R were ts, growing at 33˚C but not at 37˚C or 38˚C, while the parental virus grew at all temperatures. Using the northern blotting technique, mutant viruses N74S and S96G were shown to have a ts defect in genome-synthesis (late replication step), while mutant viruses K228T and G230R had a ts defect in antigenome- synthesis (early replication step). Host-restriction experiments were performed using 5 different cell lines (Vero-E6, BHK-21, 2FTGH-V, A549-V and 293-V). Overall, the parental virus grew similarly in all cell lines. Likewise, the majority of mutant viruses follow this pattern except mutant virus Y23A. It showed a 100-fold reduction in titer in 2FTGH-V cells. Comparing the ratios of intracellular and extracellular particles revealed that only 15% of the total virus particles of mutant Y23A was released as extracellular particles compared to 30% of the parental virus. Fourteen N genes were nonrescuable. They were characterized by (i) their activity in the BUNV minigenome system, (ii) their activity in BUNV packaging assay, (iii) their ability to form multimers, (iv) their ability to interact with L protein, and (v) their impact on RNA synthesis. In summary, BUNV N protein was shown to be multi-functional and involved in the regulation of virus transcription and replication, RNA synthesis and assembly, via interactions with the viral L polymerase, RNA backbone, itself or the viral glycoproteins.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy