Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Dissertation (Ph.D.)--University of South Florida, 2006. / Title from PDF of title page. Document formatted into pages; contains 95 pages. Includes vita. Includes bibliographical references.

Abstract In the Swedish Kingdom, of which Finland was a part until the 19th century, the parish burial records specified the cause of death earlier than other European countries. Using the information contained in 1.2 million death records from 234 different parishes, the impact of the main infectious diseases inflicted upon the Finnish population, during the period 1749–1850, is analyzed. The study is focused on the temporal and geographical distribution of three of the main epidemics; gastroenteritis, malaria and smallpox. The Industrial Revolution, the Demographic Transition and the Bacteriological Era only arrived to Finland at the end of the 19th century. The population analyzed, living at a time prior those changes, was therefore rural, scattered and with high birth and mortality rates. It was, therefore, helpless in front of many illnesses. Infectious diseases were the main cause of death, especially among children. One sixth of all analyzed deaths can be attributed to these three causes of death studied. Acute gastroenteritis generated mortality, constantly in its endemic areas (southeast of the country and Ostrobothnian coast) and also in the form of large epidemics, accompanying other factors affecting the population, such as war or famine. Smallpox was endemic in the country and caused periodic epidemic peaks which generated high mortality among children. The most densely populated areas acted as reservoirs from which the disease spread outwards the rest of the country. The generalization of the practice of vaccination, from the first decades of the 19th century, modified both the spatiotemporal distribution of the epidemics and the age distribution of smallpox mortality. Under the period studied, malaria was endemic in the southwest of Finland, being of most importance in the Åland Islands. The temporal variations in the incidence of this disease do not seem to be related to crises in the population, but rather due to environmental factors. The above causes of death greatly influenced the modulation of mortality in 18th and 19th century Finland. / Resumen En el Reino de Suecia, al que pertenecía Finlandia hasta el siglo XIX, los registros parroquiales de enterramiento especificaban la causa de muerte más tempranamente que en otros países de Europa. Utilizando la información contenida en 1,2 millones de registros de defunción, provenientes de 234 parroquias diferentes, se analiza el impacto de las enfermedades infecciosas en la población de Finlandia entre 1749 y 1850. Se estudia en especial la distribución temporal y geográfica de las mayores epidemias de gastroenteritis aguda, viruela y paludismo. Ni la revolución industrial ni la transición demográfica ni la era bacteriológica llegaron a Finlandia hasta finales del siglo XIX. La población en la época de estudio era, por tanto, rural, dispersa, con altos índices de natalidad y mortalidad y estaba indefensa ante la mayor parte de las dolencias. Las enfermedades infecciosas eran la principal causa de muerte, especialmente entre los niños. Al conjunto de las tres causas de muerte estudiadas, gastroenteritis, viruela y paludismo, puede atribuirse un sexto del total de las muertes analizadas. La gastroenteritis aguda causaba una importante mortalidad de manera constante en sus áreas endémicas (sureste del país y costa de Ostrobotnia) y una mortalidad catastrófica, en forma de grandes epidemias, en coincidencia con alteraciones en la población tales como guerras o hambrunas. La viruela era endémica en el país y se manifestaba en forma de picos epidémicos periódicos que generaban gran mortalidad entre los niños. Las zonas más densamente pobladas actuaban como reservorios desde los cuales la enfermedad se expandía al resto del país. La generalización del uso de la vacuna a partir de las primeras décadas del siglo XIX modificó tanto la distribución espaciotemporal de las epidemias como su perfil de mortalidad por edades. El paludismo era endémico en el suroeste de Finlandia durante el periodo de estudio, siendo especialmente importante en las Islas åland. Las variaciones en la importancia de esta enfermedad no parecen responder a crisis en la población sino a factores ambientales. Las causas de muerte estudiadas modularon de manera importante la mortalidad general de la Finlandia de los siglos XVIII y XIX. / Tiivistelmä Ruotsin kuningaskunnassa, johon Suomi 1800-luvulle saakka kuului, hautauksia koskevissa kirkonkirjoissa spesifioitiin kuolinsyy aikaisemmin kuin muissa Euroopan maissa. Tässä tutkimuksessa analysoidaan 234 eri kunnasta peräisin olevaa 1,2 miljoonan henkilön kuolintilastoon sisältyvää informaatiota infektiosairauksien vaikutuksesta Suomen väestöön vuosien 1749 ja 1850 välillä. Erityisesti tutkitaan akuutin mahasuolitulehduksen, isonrokon ja malarian merkittävimpien epidemioiden ajallista ja maantieteellistä levinneisyyttä. Teollinen vallankumous kuin myöskään väestöllinen muutos tai bakteriologinen aikakausi eivät saapuneet Suomeen 1800-luvun loppuun mennessä. Tutkittavan aikakauden väestö oli siten maaseudulla elävää, hajanaista, sillä oli korkea syntyvyys ja kuolleisuus ja se oli suojaton suurinta osaa sairauksia vastaan. Infektiosairaudet olivat pääasiallisin kuolinsyy, erityisesti lasten keskuudessa. Tämän kolmen tutkitun kuolinsyyn joukon, mahasuolitulehduksen, isonrokon ja malarian, voidaan katsoa aiheuttaneen kuudenneksen analysoitujen kuolemien kokonaismäärästä. Akuutti mahasuolitulehdus aiheutti tasaisesti merkittävän kuolleisuuden yleisillä esiintymisalueillaan (maan kaakkoisosa ja Pohjanmaan rannikko) ja katastrofaalisen kuolleisuuden suurten epidemioiden muodossa samanaikaisesti sellaisten väestössä tapahtuvien muutosten kuten sotien tai nälänhädän kanssa. Isorokko oli maassa yleisesti esiintyvää ja ilmeni jaksottaisten epidemiahuippujen muodossa, jotka aiheuttivat suuren kuolleisuuden lasten keskuudessa. Tiheimmin asutetut alueet toimivat reserveinä, joista sairaus levisi loppuun osaan maata. Rokotuksen käytön yleistyminen 1800-luvun ensimmäisistä vuosikymmenistä lähtien muutti niin epidemioiden alueellista ja ajallista levinneisyyttä kuin sen kuolleisuusprofiilia ikäluokittain. Malaria oli yleisesti esiintyvää Lounais-Suomessa, ja tutkittavana aikakautena se oli erityisen merkittävää Ahvenanmaan saaristossa. Muutokset tämän sairauden merkittävyydessä eivät vaikuta vastaavan väestökriisejä vaan ilmastollisia tekijöitä. Tutkitut kuolinsyyt muuttivat merkittävästi kuolleisuutta 1700- ja 1800-lukujen Suomessa.

The potential for a gastroenteritis outbreak in a post-earthquake environment may increase because of compromised infrastructure services, contaminated liquefaction (lateral spreading and surface ejecta), and the presence of gastroenteritis agents in the drinking water network. A population in a post-earthquake environment might be seriously affected by gastroenteritis because it has a short incubation period (about 10 hours). The potential for a gastroenteritis outbreak in a post-earthquake environment may increase because of compromised infrastructure services, contaminated liquefaction (lateral spreading and surface ejecta), and the presence of gastroenteritis agents in the drinking water network. A population in a post-earthquake environment might be seriously affected by gastroenteritis because it has a short incubation period (about 10 hours). The aim of this multidisciplinary research was to retrospectively analyse the gastroenteritis prevalence following the February 22, 2011 earthquake in Christchurch. The first focus was to assess whether earthquake-induced infrastructure damage, liquefaction, and gastroenteritis agents spatially explained the recorded gastroenteritis cases over the period of 35 days following the February 22, 2011 earthquake in Christchurch. The gastroenteritis agents considered in this study were Escherichia coli found in the drinking water supply (MPN/100mL) and Non-Compliant Free Associated Chlorine (FAC-NC) (less than <0.02mg/L). The second focus was the protocols that averted a gastroenteritis outbreak at three Emergency Centres (ECs): Burnside High School Emergency Centre (BEC); Cowles Stadium Emergency Centre (CEC); and Linwood High School Emergency Centre (LEC). Using a mixed-method approach, gastroenteritis point prevalence and the considered factors were quantitatively analysed. The qualitative analysis involved interviewing 30 EC staff members. The data was evaluated by adopting the Grounded Theory (GT) approach. Spatial analysis of considered factors showed that highly damaged CAUs were statistically clustered as demonstrated by Moran’s I statistic and hot spot analysis. Further modelling showed that gastroenteritis point prevalence clustering could not be fully explained by infrastructure damage alone, and other factors influenced the recorded gastroenteritis point prevalence. However, the results of this research suggest that there was a tenuous, indirect relationship between recorded gastroenteritis point prevalence and the considered factors: earthquake-induced infrastructure damage, liquefaction and FAC-NC. Two ECs were opened as part of the post-earthquake response in areas with severe infrastructure damage and liquefaction (BEC and CEC). The third EC (CEC) provided important lessons that were learnt from the previous September 4, 2010 earthquake, and implemented after the February 22, 2011 earthquake. Two types of interwoven themes identified: direct and indirect. The direct themes were preventive protocols and indirect themes included type of EC building (school or a sports stadium), and EC staff. The main limitations of the research were Modifiable Areal Units (MAUP), data detection, and memory loss. This research provides a practical method that can be adapted to assess gastroenteritis risk in a post-earthquake environment. Thus, this mixed method approach can be used in other disaster contexts to study gastroenteritis prevalence, and can serve as an appendage to the existing framework for assessing infectious diseases. Furthermore, the lessons learnt from qualitative analysis can inform the current infectious disease management plans, designed for a post-disaster response in New Zealand and internationally Using a mixed-method approach, gastroenteritis point prevalence and the considered factors were quantitatively analysed. A damage profile was created by amalgamating different types of damage for the considered factors for each Census Area Unit (CAU) in Christchurch. The damage profile enabled the application of a variety of statistical methods which included Moran’s I , Hot Spot (HS) analysis, Spearman’s Rho, and Besag–York–Mollié Model using a range of software. The qualitative analysis involved interviewing 30 EC staff members. The data was evaluated by adopting the Grounded Theory (GT) approach. Spatial analysis of considered factors showed that highly damaged CAUs were statistically clustered as demonstrated by Moran’s I statistic and hot spot analysis. Further modelling showed that gastroenteritis point prevalence clustering could not be fully explained by infrastructure damage alone, and other factors influenced the recorded gastroenteritis point prevalence. However, the results of this research suggest that there was a tenuous, indirect relationship between recorded gastroenteritis point prevalence and the considered factors: earthquake-induced infrastructure damage, liquefaction and FAC-NC. Two ECs were opened as part of the post-earthquake response in areas with severe infrastructure damage and liquefaction (BEC and CEC). The third EC (CEC) provided important lessons that were learnt from the previous September 4, 2010 earthquake, and implemented after the February 22, 2011 earthquake. The ECs were selected to represent the Christchurch area, and were situated where potential for gastroenteritis was high. BEC represented the western side of Christchurch; whilst, CEC and LEC represented the eastern side, where the potential for gastroenteritis was high according to the outputs of the quantitative spatial modelling. Qualitative analysis from the interviews at the ECs revealed that evacuees were arriving at the ECs with gastroenteritis-like symptoms. Participants believed that those symptoms did not originate at the ECs. Two types of interwoven themes identified: direct and indirect. The direct themes were preventive protocols that included prolific use of hand sanitisers; surveillance; and the services offered. Indirect themes included the EC layout, type of EC building (school or a sports stadium), and EC staff. Indirect themes governed the quality and sustainability of the direct themes implemented, which in turn averted gastroenteritis outbreaks at the ECs. The main limitations of the research were Modifiable Areal Units (MAUP), data detection, and memory loss. It was concluded that gastroenteritis point prevalence following the February 22, 2011 earthquake could not be solely explained by earthquake-induced infrastructure damage, liquefaction, and gastroenteritis causative agents alone. However, this research provides a practical method that can be adapted to assess gastroenteritis risk in a post-earthquake environment. Creating a damage profile for each CAU and using spatial data analysis can isolate vulnerable areas, and qualitative data analysis provides localised information. Thus, this mixed method approach can be used in other disaster contexts to study gastroenteritis prevalence, and can serve as an appendage to the existing framework for assessing infectious diseases. Furthermore, the lessons learnt from qualitative analysis can inform the current infectious disease management plans, designed for a post-disaster response in New Zealand and internationally.