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Three closely related isolates belonging to the A��� serotype of infectious pancreatic necrosis virus (IPNV) were selected for comparison, to provide insight into the nature of variation in the virulence of IPN viruses. Brook trout fry (Salvelinus fontinalis) were experimentally infected with the three isolates by immersion. Cumulative mortalities over a 62 day period for the three isolates were 67%, 78%, and 93%. The negative control was 3%. Virus titers from whole fish homogenates sampled at peak mortality for each isolate were statistically similar, indicating that quantity of virus does not account for virulence differences. For the two least virulent isolates, the virus titer was inversely correlated with fish weight, whereas for the most virulent isolate, no correlation was observed. Amino acid sequences of the viral capsid protein VP2 were determined using the reverse transcriptase polymerase chain reaction (RT-PCR). There were two amino acid changes at residue 217 and 288 between the two least virulent isolates and the most virulent isolate. These changes might provide a specific molecular basis for the variations in virulence among isolates. The progression of IPN virus infection in the experimentally infected fry was followed using histopathology, in situ cDNA hybridization, and alkaline phosphatase immunohistochemistry (APIH). While microscopic lesions were limited almost exclusively to necrosis of the pyloric caeca and pancreas, positive reactions with in situ hybridization and APIH were observed in tissues throughout infected fish. An IPNV infection appeared to be established in the fish by two routes: by entering the skin/lateral line and diffusing through the muscle, and from the oral region into the gastrointestinal tract by ingestion. In a second experiment, within a group of experimentally infected brook trout fry, external and behavioral signs of IPN disease in moribund fish disappeared, with the fish becoming healthy in appearance. Several of these fish were sampled, along with dead, moribund, and asymptomatic fish (never showed signs of IPN disease). Very few differences were observed among the fish sampled, using histopathology and in situ hybridization. Fish that appeared to recover after displaying signs of IPN disease died within a 2 week period. / Graduation date: 1997

The status of the Baynespruit bacteriological water quality is very alarming - E-coli concentrations have far exceeded the allowable limit of both local and international guidelines for more than a decade, namely 2000-2010. Concentrations of indicator bacteria have been recorded as high as 2419000 cfu/100 ml, whereas guideline levels of E-coli for recreational contact are about 130 cfu/100 ml. In this study, statistical analyses were carried out on data from two sampling points to clarify the seasonal changes and the variability of the pollution. Cross-correlation analyses showed that there was no significant correlation between E-coli concentrations and rainfall in the uMsunduzi catchment. There was also only a weak correlation between the two sampling points which suggests the existence of unregulated sources of pathogenic water pollution between the sampling locations that are independent of the effect that rainfall has on dilution and dispersion of pollution. The data indicates that the population living along the Baynespruit has about a 2% risk of contracting gastrointestinal illness as a result of the pollution in the stream. / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, Durban, 2011.

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology, 2012 / In many developed countries worldwide the provision of safe, clean water is an expected commodity. In South Africa however, as in most developing countries, the access and supply of water safe for human consumption is challenged or complicated by pollution and more recently water availability. Point-source pollutants in surface- and groundwater are normally the most concentrated closest to the pollutant source (such as the end of a pipe or an underground injection system). Examples of point-source pollution are commercial and industrial businesses, that often discharge waste such as solvents and heavy metals from their operations. In contrast, non-point-source pollution occurs due to runoff moving across or through the ground and absorbing and accumulating pollutants which eventually end up in streams, rivers and dams. The lack of waste removal and adequate sanitation facilities results in the disposal of faecal matter and sewage into storm water drains which flow directly into the river systems contributing to the incidence of diseases such as gastroenteritis, diarrhoea and chronic lung ailments, caused by waterborne pathogenic bacteria, viruses and fungi. Routine water quality analysis however, does not include monitoring for viral contaminants, as this process is hampered by the lack of simple, reliable, time- and cost-effective testing methods to concentrate and detect viral pathogens. The primary aim of this study was thus to establish and optimise routine monitoring techniques for the detection of rota-, adeno- and enteroviruses in the Berg- and Plankenburg Rivers, Western Cape. Initially, various concentration and extraction methods were compared for the optimum recovery of viruses from spiked water samples. One hundred milliliter water samples were spiked with one milliliter rotavirus and two milliliters adenovirus control virions (Coris Bioconcept, Gembloux, Belgium). Optimisation testing of enterovirus was however, not completed due to the unavailability of a positive control. Four viral concentration techniques, namely the Silicon dioxide (SiO2) method, positively charged, negatively charged and the mixed-ester filters, were compared. Various nucleic acid extraction methods were also employed to establish which method would provide optimum yields for both DNA and RNA nucleic acids. The extraction techniques included the TRIzol method (Invitrogen, California, USA) for RNA extraction, the Roche High Pure PCR Template Preparation kit (Roche, Mannheim, Germany) for DNA extraction, and the QIAamp Ultrasens Virus kit (Qiagen GmbH, Hilden, Germany) for simultaneous RNA and DNA extraction. The use of virus specific primers within the PCR technique was also optimised. In addition, gene specific primers and oligo(dT)15 primers were tested and compared to establish which primers would yield the best results since gene specific primers are said to be more sensitive than oligo(dT)15 primers (van Pelt-Verkuil et al., 2008) when synthesising cDNA (rotavirus). The SiO2 concentration method yielded variable results when it was used with the various nucleic acid extraction techniques in this study, since positive PCR results were obtained when used in combination III with some techniques, while negative results were obtained with others. Similarly, variable results were also obtained when negatively charged filters were used to concentrate virus particles, and when this method was used in conjunction with various virus nucleic acid extraction techniques to identify different viruses by RT-PCR and PCR. Results for the non-charged mixed-ester filter were comparable to the positively charged filters when used in conjunction with the various nucleic acid extraction techniques in this study. Both these techniques yielded the highest viral particle concentration from the spiked water samples. Pilot study results indicated the presence of rotavirus and adenovirus detected by RT-PCR and PCR respectively, when filtering through the positively charged filter. The positively charged filter/QIAamp UltraSens virus kit combination was found to be the optimum combination when analysing the spiked water results and was then employed for the concentration of virus particles in the river water samples collected from the Plankenburg- and Berg River systems throughout the study period. The expected PCR product of 346 bp for rotavirus was absent in all 72 river water samples analysed for both river systems. In contrast to the PCR results obtained for rotavirus, the expected product of 261 bp for adenovirus was detected in 22 (30.5%) samples collected throughout the study period. Fifteen of the 22 adenovirus positive samples were found in the Plankenburg River (distributed over all sites), while seven of the 22 adenovirus positive samples were found in the Berg River (all sites). A nested PCR was used to detect enterovirus in the river water samples collected from both river systems throughout the study period. In the first round of the enterovirus PCR 15 river water samples (at various sites for both river systems) yielded a faint 513 bp product. Further amplification by nested PCR then yielded 13 (18.1%) positive nested PCR products of 297 bp. The incidence of adenovirus and enterovirus in river waters reported in the current study and the Van Heerden et al. (2003) investigation motivates for similar studies to be conducted in drinking water, dam water used for recreational purposes as well as rainwater, which is gaining popularity as a sustainable water source.

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2012. / In many developed countries worldwide the provision of safe, clean water is an expected commodity. In South Africa however, as in most developing countries, the access and supply of water safe for human consumption is challenged or complicated by pollution and more recently water availability. Point-source pollutants in surface- and groundwater are normally the most concentrated closest to the pollutant source (such as the end of a pipe or an underground injection system). Examples of point-source pollution are commercial and industrial businesses, that often discharge waste such as solvents and heavy metals from their operations. In contrast, non-point-source pollution occurs due to runoff moving across or through the ground and absorbing and accumulating pollutants which eventually end up in streams, rivers and dams. The lack of waste removal and adequate sanitation facilities results in the disposal of faecal matter and sewage into storm water drains which flow directly into the river systems contributing to the incidence of diseases such as gastroenteritis, diarrhoea and chronic lung ailments, caused by waterborne pathogenic bacteria, viruses and fungi.

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) is the prototypical member of the orthopoxvirus genus of the Poxviridae family, and is currently being evaluated as a vector for vaccine development and cancer cell-targeting therapy. Despite the importance of studying poxvirus effects on the human immune system, reports of the direct interactions between poxviruses and primary human leukocytes (PHLs) are limited. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias towards binding and infecting monocytes among PHLs. VV binding strongly co-localized with lipid rafts on the surface of these cell types, even when lipid rafts were relocated to the cell uropods upon cell polarization. In humans, monocytic and professional antigen-presenting cells (APCs) have so far only been reported to exhibit abortive infections with VV. We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, were permissive to VV replication. The majority of virions produced in MDMs were extracellular enveloped virions (EEV). Visualization of infected MDMs revealed the formation of VV factories, actin tails, virion-associated branching structures and cell linkages, indicating that infected MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. Classical activation of MDMs by LPS plus IFN-γ stimulation caused no effect on VV replication, whereas alternative activation of MDMs by IL-10 or LPS plus IL-1β treatment significantly decreased VV production. The IL-10-mediated suppression of VV replication was largely due to STAT3 activation, as a STAT3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data indicate that PHL subsets express and share VV protein receptors enriched in lipid rafts. We also demonstrate that primary human macrophages are permissive to VV replication. After infection, MDMs produced EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread.