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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.

INTRODUCTION:Honey bees, Apis mellifera, have a diverse community of pathogens. Previous research has mostly focused on bacterial brood diseases of high virulence, but milder diseases caused by fungal pathogens have recently attracted more attention. This interest has been triggered by partial evidence that co-infection with multiple pathogens has the potential to accelerate honey bee mortality. In the present study we tested whether co-infection with closely related fungal brood-pathogen species that are either specialists or non-specialist results in higher host mortality than infections with a single specialist. We used a specially designed laboratory assay to expose honey bee larvae to controlled infections with spores of three Ascosphaera species: A. apis, the specialist pathogen that causes chalkbrood disease in honey bees, A. proliperda, a specialist pathogen that causes chalkbrood disease in solitary bees, and A. atra, a saprophytic fungus growing typically on pollen brood-provision masses of solitary bees.RESULTS:We show for the first time that single infection with a pollen fungus A. atra may induce some mortality and that co-infection with A. atra and A. apis resulted in higher mortality of honey bees compared to single infections with A. apis. However, similar single and mixed infections with A. proliperda did not increase brood mortality.CONCLUSION:Our results show that co-infection with a closely related fungal species can either increase or have no effect on host mortality, depending on the identity of the second species. Together with other studies suggesting that multiple interacting pathogens may be contributing to worldwide honey bee health declines, our results highlight the importance of studying effects of multiple infections, even when all interacting species are not known to be specialist pathogens.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Chronic inflammation and immune activation are hallmarks of HIV infection, resulting in chronic oxidative stress with over-utilization of antioxidant defences, which may contribute to the loss of immune cells and faster disease progression. Low levels of antioxidants in HIV- infected individuals have been associated with frequent opportunistic infections and an increased risk of mortality. HIV infection is also associated with on-going and aberrant activation of both the innate and adaptive immune systems. An important aspect of innate immune stimulation is derived from the leakage of lipopolysaccharide (LPS) across the damaged mucosal lining of the gut in early HIV infection. The impact of this innate immune stimulation on the adaptive arm of the immune system, as represented in this study by levels of CD4+ T-cell activation and death, have not been explored previously in untreated HIV infection. Using an integrated approach of immune activation, inflammation, oxidative stress and ameliorating antioxidant intervention for the first time, this thesis reports the impact of inflammatory induced-oxidative stress on CD4+ T-cells in an in vitro HIV model. In a preliminary study, baseline levels of neutrophil respiratory burst as an in vitro indication of immune stimulation were investigated. The relationships between baseline total antioxidant status (TAS), Red blood cell (RBC) antioxidant enzyme activities (catalase, superoxide dismutase & glutathione peroxidase), lipid peroxidation and glutathione redox ratio and other markers of disease in asymptomatic, untreated HIV infection were also explored. The design and optimization of an assay that could determine the effects of LPS-induced oxidative stress on CD4+ T-cells, was a critical part of this study. The development of this assay enabled the measurement of the effects of selected antioxidant interventions N-acetyl cysteine (NAC) and vitamin C, on LPS-induced CD4+ T-cell activation and death. The results were also correlated with CD4 count, viral load and markers of inflammation (fibrinogen & D-dimers) in HIV-infected and uninfected groups. Neutrophils from HIV-infected persons at rest showed a ―primed‖ response to low stimulating agent, bacterial N-formyl peptides (fMLP), which was significantly (P = 0.04) higher than uninfected individuals. There was increased oxidative stress as evidenced by increased catalase activity, malondialdehyde (MDA) and conjugated dienes (CDs) with a corresponding decrease in antioxidant capacity in HIV-infected individuals with lower CD4 count. NAC in combination with vitamin C, significantly (P = 0.0018) reduced activation of CD4+ T-cells to a greater degree than with either antioxidant alone. NAC and vitamin C individually and in combination significantly (P = 0.05, P = 0.012 and P<0.0001) decreased the expression of the markers of apoptosis, Annexin V and 7-amino-actinomycin (7-AAD). Importantly, the antioxidant combination decreased MDA values and significantly (P = 0.01) increased the glutathione redox ratio in the HIV-infected group. Based on these results, the respiratory burst and LPS-induced activation may be important contributing factors in inflammatory-associated oxidative stress in HIV infection and contribute to the depletion of CD4+ T-cells in the asymptomatic stage of HIV infection. These results also indicate the potential inhibitory effects of NAC and vitamin C in combination as agents that may limit immune activation and inflammation-induced oxidative stress. Importantly, the study showed that at this asymptomatic stage, CD4+ T-cells of the HIV-infected group responded similarly to stimulation as the HIV negative group, indicating that antioxidant defences were still functional and that appropriate early intervention at this stage may be protective against oxidative damage to the immune cells. To the best of our knowledge, this study is the first to use an integrated approach involving not only plasma levels of antioxidant status, but also RBC antioxidant enzyme activities, oxidative damage (lipid peroxidation), inflammation, cellular levels of immune activation and potential ameliorating interventions in evaluating the problem of inflammation-induced oxidative stress in HIV infection. Based on the results of this study, it is envisaged that an insight into the immune activation, inflammatory and oxidative stress status of patients will enable long-term profiling of each patient with a view to individualized therapy. This approach may have a direct impact on patient care in resource-limited settings such as sub-Saharan Africa. / AFRIKAANSE OPSOMMING: Chroniese inflammasie en immuunaktivering is kenmerke van MIV-infeksie. Dié twee prosesse lei tot chroniese oksidatiewe stres en oorbenutting van antioksidant verdedigingstelsels, wat lei tot die verlies van die immuun selle en vinniger siektevordering. Lae vlakke van antioksidante in MIV-positiewe individue stem ooreen met gereelde opportunistiese infeksies en 'n verhoogde risiko van mortaliteit. MIV-infeksie word ook geassosieer met langdurige en abnormale aktivering van beide die ingebore en aanpasbare immuunstelsels. 'n Belangrike aspek van ingebore immuun stimulasie in die raamwerk van vroeë MIV-infeksie, is die lekkasie van LPS oor die beskadigde slymvlies voering van die dunderm. Die impak van die ingebore immuun stimulasie op die aanpasbare arm van die immuunstelsel, soos aangetoon in hierdie studie deur die vlakke van CD4 T-sel aktivering en apoptose, is nog nie voorheen ondersoek in onbehandelde MIV-infeksie nie. Met behulp van 'n oorspronklike, geïntegreerde benadering van immuun aktivering, inflammasie, oksidatiewe stres en 'n lae vlak van antioksidant intervensie, lewer hierdie tesis verslag oor 'n in vitro model van inflammasie-geïnduseerde oksidatiewe stres op CD4 T-selle. In 'n voorlopige studie, is basislyn vlakke van die neutrofiel respiratoriese uitbarsting as 'n in vitro aanduiding van immuunstimulasie ondersoek. Die verhoudinge tussen basislyn totale antioksidant status (TAS), rooi bloed sel (RBC) antioksidant ensiemaktiwiteit (katalase, superoksied dismutase, en glutatioon peroksidase), lipied peroksidasie en glutatioon redoks-verhouding, asook ander merkers van siektevordering in asimptomatiese, onbehandelde MIV-infeksie is ook ondersoek. Die ontwerp en optimisering van 'n toets wat die effek van LPS-geïnduseerde oksidatiewe stres op CD4 T-selle kan bepaal, was 'n kritieke deel van hierdie studie. Die ontwikkeling van hierdie toets het ook die meting van die effek van toevoeging van twee geselekteerde anti-oksidante, N-asetiel sisteïen (NAC) en vitamien C, op LPS-geïnduseerde CD4 T-sel aktivering en apoptose ondersoek. Die resultate is ook gekorreleer met CD4-telling, virale lading en merkers van inflammasie (fibrinogeen en D-dimere) in groepe met en sonder MIV-infeksie. Neutrofiele van asimptomatiese persone met MIV infeksie, het 'n 'voorbereide' reaksie gehad teen ‗n lae stimulerende agent, bakteriële N-formiel peptied (fMLP), wat beduidend (P = 0,04) hoër was as in individue sonder MIV infeksie. Daar was verhoogde oksidatiewe stres soos bewys deur verhoogde katalase aktiwiteit, malondialdehied (MDA) en gekonjugeerde diëne (CDs), saam met 'n ooreenstemmende afname in anti-oksidant kapasiteit in MIV-positiewe individue met laer CD4-tellings. NAC in kombinasie met vitamien C, het die aktivering van CD4 T-selle beduidend verminder (P = 0,0018), 'n effek wat groter was in vergelyking met elke antioksidant alleen. NAC en vitamien C alleen, en in kombinasie het beduidend die uitdrukking van die merkers van apoptose, Annexin V en 7-AAD verminder (P = 0,05, P = 0.012 en P<0,0001). Wat belangrik is, is dat die afname in MDA waardes as gevolg van antioksidante in kombinasie, 'n beduidende styging in die glutatioon redoks verhouding in die MIV-positiewe groep tot gevolg gehad het. Hierdie resultate het aangetoon dat die respiratoriese uitbarsting en LPS-geïnduseerde aktivering belangrike bydraende faktore mag wees in inflammasie-verwante oksidatiewe stres in MIV-infeksie, wat kan bydra tot die uitputting van CD4 T-selle in die asimptomatiese stadium van MIV-infeksie. Hierdie resultate dui ook aan dat die moontlike inhiberende effekte van NAC en vitamien C in kombinasie, immuun aktivering en geïnduseerde oksidatiewe stres kan beperk. Van belang is die feit dat hierdie studie bewys het dat in die asimptomatiese stadium van MIV-infeksie, CD4 T-selle weens stimulasie dieselfde gereageer het as dié van mense sonder MIV infeksie. Dit het aangedui dat antioksidant verdediging in hierdie stadium nog funksioneel was, en dat 'n toepaslike vroeë intervensie op hierdie stadium beskermend teen immuun-sel oksidatiewe skade kan wees. Tot die beste van ons kennis, is hierdie studie die eerste om 'n geïntegreerde benadering te gebruik, waar plasma vlakke van antioksidant status saam met RBC antioksidant ensiemaktiwiteit, oksidatiewe skade (lipied peroksiidasie), inflammasie, sellulêre vlakke van immuunaktivering, en potensiële beskermende ingrypings ondersoek is in die evaluering van die probleem van oksidatiewe stres in MIV-infeksie wat tot inflammasie lei. Gebaseer op dié resultate, word dit in die vooruitsig gestel dat 'n insig in die status van immuunaktivering, inflammasie, en oksidatiewe stress van pasiënte, dit moontlik sal maak vir langtermyn- beplanning om vir elke pasiënt individuele terapie voor te skryf. Hierdie benadering kan 'n direkte impak op die sorg van pasiënte in hulpbron-beperkte gebiede soos sub-Sahara Afrika hê.

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

published_or_final_version / Community Medicine / Master / Master of Public Health

published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Clostridium difficile infection (CDI) is the leading cause of bacterial infectious diarrhea in nosocomial settings and approximately 25% of patients with CDI experience disease recurrence. Prior CDI epidemiological investigations are limited though. They do not reflect the burden of CDI in federal facilities, nor do they capture recent estimates on patient health outcomes. Furthermore, few studies have integrated CDI recurrence risk factors into a tool that clinicians can use to identify patients at risk for CDI recurrence. This study 1) described the epidemiology of CDI in the national Veterans Health Administration (VHA), 2) derived and validated a clinical prediction rule for 60-day CDI first recurrence, and 3) derived and validated a clinical prediction rule for 60-day CDI second recurrence. This was a retrospective cohort study of VHA beneficiaries with CDI between October 1, 2001 and September 30, 2012. VHA clinical and pharmacy data were integrated to develop several independent variables, including patient baseline demographics, CDI characteristics, comorbidities, concomitant medications, prior medications, prior hospitalization, hospital length of stay (LOS), and CDI severity. The dependent variables included 30/60/90-day mortality, and 30/60/90-day CDI recurrence. CDI incidence and outcomes were presented descriptively and compared using generalized linear regression models. CDI recurrence prediction rules were derived using multivariable logistic regression models and validated using the area under the receiver-operating-characteristic curve (AUROC), sensitivity, specificity, positive predictive value, and negative predictive value. Our study demonstrated that CDI first episodes, recurrences, and severity increased over the study period, while mortality decreased. Our CDI first recurrence prediction rule included the following predictor variables: dyslipidemia, CDI type, renal disease, hospital LOS <7 days, principal CDI diagnosis, concomitant gastric acid suppressors, and concomitant antibiotics. This model demonstrated moderate 60-day first recurrence discrimination (AUROC=0.62). Our CDI second recurrence prediction rule was similar in predictor variables and validity. In conclusion, CDI is an important, rapidly-emerging public health problem in the VHA. A clinical prediction rule might aid clinicians in directing preventative therapies to patients at high risk for CDI recurrence. / text

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