No description available.

African horsesickness virus (AHSV) belongs to the Orbivirus genus within the family Reoviridae. AHSV is transmitted to vertebrates by Culicoides midges and causes an acute disease in horses with a high mortality rate. The virion consists of an outer layer composed of proteins VP2 and VP5, which surround an icosahedral core containing two major proteins, VP3 and VP7, three minor proteins, VP1, VP4 and VP6, and ten segments of double-stranded (ds)RNA. The VP7 protein is not only important in maintaining the structural integrity of the virus particles, but has been reported to play a key role in Culicoides cell entry. The phenomenon of RNA interference (RNAi), which can be used to selectively silence homologous genes post-transcriptionally, has revolutionized approaches to study gene function and it is also projected as a potential tool to inhibit virus replication. In mammalian cells, RNAi can be triggered by the direct introduction of 21-23 nucleotide duplexes of small interfering RNA (siRNA) that specifically and potently inhibits endogenous and heterogeneous gene expression. Consequently, the aims of the investigation were to develop RNAi assays whereby the expression of the AHSV-9 VP7 gene could be suppressed in Spodoptera frugiperda insect cells and in mammalian BHK-21 cell culture, as well as to determine whether gene-specific siRNAs may prevent AHSV-9 infection in cell culture. To investigate RNAi-mediated silencing of an enhanced green fluorescent protein (eGFP) in S.frugiperda cells, the bacmid expression system was used. Transfection of the insect cells with an eGFP-specific siRNA prior to infection with the recombinant bacmid, inhibited eGFP protein expression by 75%, as quantified by fluorometry. Although the results suggest that RNAi could potentially be used as tool to study the function of an expressed transgene in insect cells, the lack of complete inhibition, coupled with the highly cytolytic nature of the bacmid, may complicate interpretation of the gene interference results. To investigate whether siRNAs targeting the AHSV-9 VP7 mRNA is able to silence VP7 protein expression, two siRNAs were designed that targeted different regions on the VP7 mRNA. siVP7-336 and siVP7-441, directed at nucleotides 336-356 and 441-461 on the VP7 coding strand, respectively, were chemically synthesized. The effect of these siRNAs on VP7 protein expression was evaluated by cotransfection of BHK-21 cells with the respective siRNAs and the VP7 expression plasmid pCMVVP7- eGFP. The results indicated that siVP7-336 and siVP7-441 inhibited VP7-eGFP expression by 88% and 75%, respectively. BHK-21 cells were subsequently transfected with the respective siRNAs in separate experiments followed by viral infection. The VP7 mRNA quantities were measured by quantitative reverse transcription PCR and the effect of the siRNAs on viral replication was evaluated by plaque assays. Of the two siRNAs, siVP7-336 was found to be the most effective inhibitor of VP7 transcription and suppressed VP7 mRNA by 93%. The exposure of BHK-21 cells to the VP7 genespecific siRNA, siVP7-336, also led to a 84% reduction in progeny virions, as measured by a plaque assay. Taken together, the results demonstrate that siRNA-mediated gene silencing is an efficient approach for reducing the level of VP7 transcripts and proteins and for inhibiting virus propagation. / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Rotaviruses are the single most common cause of fatal and severe childhood diarrhoeal illness worldwide (>125 million cases annually). Rotavirus shares structural and functional features with many viruses, such as the presence of segmented double-stranded RNA genomes selectively and tightly packed with a conserved number of transcription complexes in icosahedral capsids. Nascent transcripts exit the capsid through 12 channels, but it is unknown whether these channels specialise in specific transcripts or simply act as general exit conduits; a detailed description of this process is needed for understanding viral replication and genomic organisation. To test these opposing models, a novel single-molecule assay was developed for the capture and identification (CID) of newly synthesised specific RNA transcripts. CID combines the hybridisation of transcripts with biotinylated and FRET compatible labelled ssDNAs with the implementation of recent developments in single molecule fluorescence such as alternating laser excitation (ALEX) and total internal reflection fluorescence (TIRF) microscopy. CID identifies and quantifies specific transcripts of rotavirus based on a FRET/Stoichiometry (E*/S) value of the hybridised labelled probes. I used CID to pull down the capsid on the surface slide and identify partially extruded transcripts of three different segments 2, 6 and 11. The findings presented in this thesis support a model in which each channel specialises in extruding transcripts of a specific segment, that in turn is linked to a single transcription complex. The method can be extended to study other transcription systems including E.coli, and can be further developed as a potential diagnostic tool.

No description available.

African horse sickness (AHS) is a life threatening disease of equids caused by African horse sickness virus (AHSV), a member of the genus Orbivirus in the family Reoviridae. The virus is transmitted to horses by midges (Culicoides spp.) and the disease is most prevalent during the time of year, and in areas where the Culicoides spp. are most abundant, namely in late summer in the summer rainfall areas of the country. Whilst the clinical signs and presentation of the disease were well documented by Sir Arnold Theiler (1921), very little is known or documented about AHSV dynamics or the clinical pathological and serological responses of horses to natural infection with AHSV. This dissertation describes the history and current knowledge on AHS, and the methods and results of a prospective study on natural AHSV infection of horses, undertaken between 2009 and 2010 by the Equine Research Centre (ERC) at the University of Pretoria, Faculty of Veterinary Science, Onderstepoort. This study is the first documented study of its nature and included animals of various ages and therefore variable vaccination status. The objectives of the study were to describe the viral dynamics of AHSV infection in horses, to gain a better understanding of the clinical pathological and serological responses to natural AHS infection and to demonstrate early detection of AHS infection in horses under field conditions. / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

Reovirus is internalized after interaction of the outer proteins μ1, σ1 and σ3 with the host cell. Proteolysis of σ3 and cleavage of μ1 (into δ and φ) eventually leads to the formation of a more infectious subviral particle named “ISVP”. The infectious entry of viruses, but not of ISVPs, can be blocked using various entry inhibitors and therefore, suggests that there is a threshold of σ3 digestion required to allow particle to bypass entry blockers. By combining protease and detergent to the digestion of virions, data from this work showed distinct particles generated along the transition pathway. In addition, studies involving flow cytometry and specific antibodies (anti-μ1) showed that between virus and ISVP there is a gradual yet heterogeneous particle proteolysis that is directly related to the virus infectivity. The findings and approaches taken for this thesis work can possibly be extended for studying other non-enveloped viruses. Moreover, it may help to shed some light on the development of safe and effective oncolytic agents.

Reovirus is internalized after interaction of the outer proteins μ1, σ1 and σ3 with the host cell. Proteolysis of σ3 and cleavage of μ1 (into δ and φ) eventually leads to the formation of a more infectious subviral particle named “ISVP”. The infectious entry of viruses, but not of ISVPs, can be blocked using various entry inhibitors and therefore, suggests that there is a threshold of σ3 digestion required to allow particle to bypass entry blockers. By combining protease and detergent to the digestion of virions, data from this work showed distinct particles generated along the transition pathway. In addition, studies involving flow cytometry and specific antibodies (anti-μ1) showed that between virus and ISVP there is a gradual yet heterogeneous particle proteolysis that is directly related to the virus infectivity. The findings and approaches taken for this thesis work can possibly be extended for studying other non-enveloped viruses. Moreover, it may help to shed some light on the development of safe and effective oncolytic agents.

Thesis (Ph. D.)--West Virginia University, 2005 / Title from document title page. Document formatted into pages; contains ix, 155 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xi, 288 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains x, 202 p. : ill. (some col.). Includes abstract. Includes bibliographical references.