Thesis (PhD (Medical Virology))-- University of Limpopo, 2010. / Background: Rotavirus infection remains a significant clinical problem throughout the world, infecting almost every child younger than 5 years of age, despite socio-economic status or environmental conditions. Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants and young children. Implementation of an effective vaccine programme could reduce the incidence and severity of rotavirus disease. Decisions about new candidate rotavirus vaccines require reliable data on disease impact in both developed and developing countries. The aim of this study was to assess the burden of rotavirus associated disease at the tertiary care Dr George Mukhari Hospital, Ga-Rankuwa and the secondary care hospital Brits Hospital, Madibeng and to describe the genetic diversity of rotavirus strains circulating in Ga-Rankuwa and Brits communities over a similar time period as the testing of Rotarix@ vaccine. The broad objectives included; to perform a hospital-based burden of rotavirus disease in two different hospitals in the North West of Pretoria area, to conduct molecular characterization of rotaviruses circulating in the Pretoria region and lastly to devise an alternative molecular typing method to detect rotavirus VP6 subgroups. Materials and Method: To investigate the hospital-based burden of rotavirus disease, diarrhoeal stool samples were collected at Dr George Mukhari and Brits Hospitals from children less than 5 years of age. Group A rotavirus antigen was detected from the samples using commercially available rotavirus enzyme immunoassay IDEIA TM Rotavirus test (DAKO, Dakocytomation, Denmark). Genetic analyses of rotavirus strains were determined by polyacrylamide gel electrophoresis (PAGE) to characterize the electrophoretic patterns followed by analysis of the P and G genotypes by RT -PCR and multiplex PCR amplification of specific sequences of VP7 and VP4 genes. To devise an alternative molecular typing method to detect rotavirus VP6 subgroups, with subgroup specificities determined by both VP6 monoclonal antibodies and restriction fragment length polymorphism using restriction endonuclease Acil, Odel and Rsa I. Selected PCR amplicons (VP7 and VP6 genes) were purified, cloned and sequenced. Consensus sequences of the VP7 and VP6 genes were aligned and analysed manually with Chromaslite and BioEdit software packages. Multiple sequence alignment was implemented by Mafft software packages. The nucleotide and deduced amino acid sequences of the VP7 and VP6 genes were compared with reference strains available from GenBank. Multiple methods were used to construct phylogenetic trees and included neighbor¬ joining, maximum parsimony analysis and maximum likelihood distance. Bootstrap values were computed using 1000 replicates with Phylip and the MEGA softwares. The graphic representation of each phylogenetic tree was displayed with the Treeview program. Results: Between 2003 and 2005, a total of 2 514 diarrhoeal stool samples were collected. Of these, 527 (21%) were positive for group A rotavirus and the majority of children hospitalized were less than 2 years of age. The annual peak prevalences of group A rotavirus were 56%, 59% and 56% for 2003, 2004 and 2005, respectively and were observed during the autumn and winter months. The estimated incidence of gastroenteritis associated with rotavirus indicates that one in every 50 to 70 children in the area is likely to be hospitalized with rotavirus diarrhoea between birth and 2 years of age. During the three-year study period, ten, six and seven different RNA electrophoretic patterns were identified in 2003, 2004 and 2005, respectively. The VP6 genes of the representative strains (G1, G2, G3, G9, G8 and G12) were ana lysed with restriction endonuclease Acil, Ode! and Rsa!. The restriction endonucleases produced 11 unique restriction profiles (A-K). The VP6 RFLP results correlated well with strains displaying long RNA electropherotypes and VP6 subgroup 1/ specificity and also with strains displaying short RNA electropherotypes and exhibiting VP6 subgroup I specificity as determined with VP6 monoclonal antibodies. The genotypic distribution varied remarkably and major rotavirus strains detected in circulation during the study period included G2P[4] in 2003, G1 P[8] in 2004 and G3P[8]/ G3P[6] in 2005. Rotavirus strains carrying G8P[8] specificities and unusual G 12P[6] strains were also detected at low frequency The consensus VP7 nucleotide sequences, exhibited the greatest homology and identity (97-99%), when compared against corresponding international reference strains. The nucleotide sequence datasets were closely related to strains from South Africa, Vietnam, Bangladesh, East India, Republic of Congo, China, Russia, Thailand and Japan. The phylogenetic tree revealed the South African strains (G1-G3, G8-G9 and G12) clustered with international strains whereas the G1 strains clustered into two different lineages. Phylogenetic analysis of the VP6 gene revealed four lineages with international reference strains. The VP6 gene showed 97-99% identity at the deduced amino acids level with strains from Taiwan, Bangladesh, the United States and Brazil. Conclusion: This is the first study to estimate the disease burden associated with rotavirus diarrhoea in South Africa. The overall results confirm that rotavirus is the most common cause of severe diarrhoea. The epidemiology of rotavirus diarrhoea in South Africa correlates well with what has been reported in other countries. The proportion of hospitalization of rotavirus infection in children less than 5 years was estimated to an annual prevalence of 22.8% (95%CI 21.2%, 24.5%) at Dr George Mukhari Hospital, while at Brits Hospital was estimated at 18.2% (95%CI 14.9%, 22.1 %). Rotavirus genotypes circulating at Dr George Mukhari Hospital showed a high degree of diversity and the emergence of uncommon rotavirus strains such as G12. The emergence of novel rotaviruses in the region needs to be taken into account where vaccine efficacy is concerned. It is, thus, important to continue with such studies to monitor the rotavirus strains associated with severe gastroenteritis in a hospital setting before and after the introduction of a rotavirus vaccine. Results also indicated that RFLP analysis of VP6 amplicons might be a simple and reliable, alternative to MAb subgrouping. The sequence analysis of the partial length VP6 gene confirmed the location and the recognition sites of the restriction enzymes The RFLP analysis proved to have more potential to accurately detect different rotavirus subgroups.

Thèse d'exercice : Médecine. Pédiatrie : Paris 12 : 2005. / Titre provenant de l'écran-titre. Bibliogr. f. 60-66.

Thesis (Ph. D.)--University of Iowa, 2008. / Thesis supervisors: Herbert W. Hethcote, Tong Li. Includes bibliographical references (leaves 105-110).

Objetivo: Comparar la gravedad de casos con enfermedad diarreica aguda en niños menores de cinco años con y sin rotavirus. Material y Métodos: Estudio descriptivo observacional retrospectivo en una muestra de 119 niños menores de 5 años con diarrea aguda, atendidos en un Hospital Centinela de Lima durante el año 2013. El diagnóstico de rotavirus se realizó a través del test inmunocromatografíco ELISA, para determinar la severidad de la enfermedad se utilizó el score de Vesikari. Los resultados fueron expresados en cifras absolutas y relativas, el análisis se realizó a través de medidas de tendencia central, χ2 y odds ratios con sus respectivos intervalos de confianza. Resultados: En 30 niños menores de 5 años (25.2%) se identificó rotavirus, a predominio de mujeres de 12 a 23 meses de edad. Se encontró diferencias significativas entre el grupo de rotavirus positivo y negativo en el número de diarreas en las últimas 24 horas, la media de días de vómitos, en el número de vómitos en las últimas 24 horas, en el grado de deshidratación, el tratamiento recibido y en el score de Vesikari. Conclusiones: Los resultados muestran que existen diferencias clínicas y mayor gravedad de la diarrea por rotavirus en niños menores de 5 años en relación con niños con rotavirus negativo.

Thesis (MSc (Medical science in medical virology)) -- University of Limpopo / Background: It has been established that rotaviruses are the main cause of acute gastroenteritis in children worldwide, resulting in more than 453 000 deaths, with a high mortality still occurring in African countries and Asia. In Swaziland, diarrheal diseases are a common cause of morbidity and mortality among children <5 years of age. Approximately 10% of hospitalised Swazi children die due to diarrhoea every year. Through financial assistance from the World Health Organization (WHO), many African countries have conducted a lot of rotavirus disease studies. In Swaziland, the epidemiology of rotavirus infection is unknown due to lack of data. Thus, the study’s aim was to examine the epidemiology and characterize rotavirus strains in children <5 years of age, hospitalised and attending the outpatient departments of public and private healthcare facilities in Swaziland. Materials and methods: A total of 745 diarrheal stool specimens were collected from children <5 years of age from April 2009 to December 2010. Group A rotavirus antigen was detected using a commercially available enzyme immunoassay (EIA) kit (ProSpectTM, Oxoid Ltd, UK). Polyacrylamide gel electrophoresis (PAGE) was used to determine the electrophoretic pattern of rotavirus strains. The P and G genotypes were established by reverse transcription polymerase chain reaction (RT-PCR) and multiplex hemi-nested PCR amplification of the VP4 and VP7 genes respectively, using type-specific primers. Sequencing was performed on 35 specimens to confirm the circulating genotypes. The phylogenetic tree and similarity distances between genotypes were constructed using the neighbour joining method and the Kimura two-parameter model package in the MEGA version 5.05 software program. Results: Group A rotavirus was detected at 13.3% in 2009 (based on samples collected from April to December) and 23.4% in 2010 (based on one year collection) from children <5 years of age hospitalized and attending outpatient departments. The rotavirus infection was more frequently detected in the age group 0-11 months (22.2%). Gender did not play a major role in rotavirus infection, because both male (20.8%) and female (18.8%) children were equally affected. Of the children that were admitted in the hospital, 33.3% were affected by rotavirus infection compared to those attending the outpatient departments (13.5%). The rotavirus infection was observed during the cooler, drier months of the year. The three most predominant G and P genotypes detected were G2P[4] (30.4%), followed by G1P[8] (15.5%) and G9P[8] (8.8%). A significant number of uncommon rotavirus strains (32.4%), mixed infections (8.8%) and nontypeables (4.1%) were also detected. The circulating genotypes detected were classified into lineages and sub-lineages defined by phylogenetic analysis of nucleotide sequences. The Swaziland strains were found clustering with known African and global strains from the GenBank. Conclusion: The findings of this study reveal that group A rotaviruses are the etiological agents of severe diarrhoea in children under 5 years in Swaziland. The diversity of rotavirus strains that were detected highlights the importance of introducing the rotavirus vaccine in the country. The currently licensed vaccines may confer protection against the circulating strains detected in this study. Data on the burden of rotavirus disease in Swaziland will be used to convince the Ministry of Health and policy makers in the country to advocate for the introduction of the rotavirus vaccine. This is the first data on the epidemiology and characterization of rotavirus strains in Swaziland; therefore there is a need for continuing with the surveillance of rotavirus in the existing sentinel sites to determine the impact of rotavirus infection over time. It is also essential to continuously monitor the rotavirus strains circulating among Swazi children.

Rotavirus non structural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum intracellular receptor. Continued studies of NSP4 revealed additional functions performed by or dependent on NSP4, some of which required trafficking from the ER to other areas of the cell. Chiefly, purified NSP4 exogenously added to the PM has been shown to mobilize intracellular calcium by a phospholipase C/inositol trisphosphate signaling pathway, yet the details whereby NSP4 are able to exert enterotoxic actions are still unknown. Our initial hypothesis included the protein caveolin 1, which subsequently was proven to bind NSP4 and prompted continued investigation as to whether or not NSP4 utilized caveolin 1 for extrareticular transport and or function. Caveolin 1 is the defining protein of caveolae, a region of the plasma membrane rich in multiple molecules that function in signal transduction, including possible receptor mediated activation of the phospholipase C/inositol triphosphate pathway. To determine if NSP4 trafficked to caveolae, a novel isolation procedure was developed and utilized to show NSP4 in PM caveolae. Expanding on the caveolae/NSP4 finding, temporal and spatial analyses of NSP4 in relation to progeny virus were conducted. NSP4's appearance at the exofacial surface of the PM was carried out utilizing surface biotinylation, exofacial staining of live cells, and confocal imaging of the PM with fluorescent resonance energy transfer studies. During these studies soluble NSP4, which was isolated from RV infected cells, was also shown to interact with the PM of multiple cell lines. These studies provided confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins. Our studies indicate the presence of full length NSP4 glycans at caveolae and the exofacial PM and are in agreement with studies indicating NSP4 traffics independent of the Golgi network. To further explore the NSP4/caveolin 1 interaction, we conducted a comparative analysis of NSP4 in relation to two separate pools of proteins. The first pool included proteins collocated with the classical secretory pathway proteins, including caveolin 1, which traffick through the Golgi. The second pool included proteins collocated with a subset of caveolin 1, which traffick independently of the Golgi.

Rotavirus non structural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum intracellular receptor. Continued studies of NSP4 revealed additional functions performed by or dependent on NSP4, some of which required trafficking from the ER to other areas of the cell. Chiefly, purified NSP4 exogenously added to the PM has been shown to mobilize intracellular calcium by a phospholipase C/inositol trisphosphate signaling pathway, yet the details whereby NSP4 are able to exert enterotoxic actions are still unknown. Our initial hypothesis included the protein caveolin 1, which subsequently was proven to bind NSP4 and prompted continued investigation as to whether or not NSP4 utilized caveolin 1 for extrareticular transport and or function. Caveolin 1 is the defining protein of caveolae, a region of the plasma membrane rich in multiple molecules that function in signal transduction, including possible receptor mediated activation of the phospholipase C/inositol triphosphate pathway. To determine if NSP4 trafficked to caveolae, a novel isolation procedure was developed and utilized to show NSP4 in PM caveolae. Expanding on the caveolae/NSP4 finding, temporal and spatial analyses of NSP4 in relation to progeny virus were conducted. NSP4’s appearance at the exofacial surface of the PM was carried out utilizing surface biotinylation, exofacial staining of live cells, and confocal imaging of the PM with fluorescent resonance energy transfer studies. During these studies soluble NSP4, which was isolated from RV infected cells, was also shown to interact with the PM of multiple cell lines. These studies provided confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins. Our studies indicate the presence of full length NSP4 glycans at caveolae and the exofacial PM and are in agreement with studies indicating NSP4 traffics independent of the Golgi network. To further explore the NSP4/caveolin 1 interaction, we conducted a comparative analysis of NSP4 in relation to two separate pools of proteins. The first pool included proteins collocated with the classical secretory pathway proteins, including caveolin 1, which traffick through the Golgi. The second pool included proteins collocated with a subset of caveolin 1, which traffick independently of the Golgi.

Rotavirus non structural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum intracellular receptor. Continued studies of NSP4 revealed additional functions performed by or dependent on NSP4, some of which required trafficking from the ER to other areas of the cell. Chiefly, purified NSP4 exogenously added to the PM has been shown to mobilize intracellular calcium by a phospholipase C/inositol trisphosphate signaling pathway, yet the details whereby NSP4 are able to exert enterotoxic actions are still unknown. Our initial hypothesis included the protein caveolin 1, which subsequently was proven to bind NSP4 and prompted continued investigation as to whether or not NSP4 utilized caveolin 1 for extrareticular transport and or function. Caveolin 1 is the defining protein of caveolae, a region of the plasma membrane rich in multiple molecules that function in signal transduction, including possible receptor mediated activation of the phospholipase C/inositol triphosphate pathway. To determine if NSP4 trafficked to caveolae, a novel isolation procedure was developed and utilized to show NSP4 in PM caveolae. Expanding on the caveolae/NSP4 finding, temporal and spatial analyses of NSP4 in relation to progeny virus were conducted. NSP4’s appearance at the exofacial surface of the PM was carried out utilizing surface biotinylation, exofacial staining of live cells, and confocal imaging of the PM with fluorescent resonance energy transfer studies. During these studies soluble NSP4, which was isolated from RV infected cells, was also shown to interact with the PM of multiple cell lines. These studies provided confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins. Our studies indicate the presence of full length NSP4 glycans at caveolae and the exofacial PM and are in agreement with studies indicating NSP4 traffics independent of the Golgi network. To further explore the NSP4/caveolin 1 interaction, we conducted a comparative analysis of NSP4 in relation to two separate pools of proteins. The first pool included proteins collocated with the classical secretory pathway proteins, including caveolin 1, which traffick through the Golgi. The second pool included proteins collocated with a subset of caveolin 1, which traffick independently of the Golgi.

Rotavirus non structural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum intracellular receptor. Continued studies of NSP4 revealed additional functions performed by or dependent on NSP4, some of which required trafficking from the ER to other areas of the cell. Chiefly, purified NSP4 exogenously added to the PM has been shown to mobilize intracellular calcium by a phospholipase C/inositol trisphosphate signaling pathway, yet the details whereby NSP4 are able to exert enterotoxic actions are still unknown. Our initial hypothesis included the protein caveolin 1, which subsequently was proven to bind NSP4 and prompted continued investigation as to whether or not NSP4 utilized caveolin 1 for extrareticular transport and or function. Caveolin 1 is the defining protein of caveolae, a region of the plasma membrane rich in multiple molecules that function in signal transduction, including possible receptor mediated activation of the phospholipase C/inositol triphosphate pathway. To determine if NSP4 trafficked to caveolae, a novel isolation procedure was developed and utilized to show NSP4 in PM caveolae. Expanding on the caveolae/NSP4 finding, temporal and spatial analyses of NSP4 in relation to progeny virus were conducted. NSP4's appearance at the exofacial surface of the PM was carried out utilizing surface biotinylation, exofacial staining of live cells, and confocal imaging of the PM with fluorescent resonance energy transfer studies. During these studies soluble NSP4, which was isolated from RV infected cells, was also shown to interact with the PM of multiple cell lines. These studies provided confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins. Our studies indicate the presence of full length NSP4 glycans at caveolae and the exofacial PM and are in agreement with studies indicating NSP4 traffics independent of the Golgi network. To further explore the NSP4/caveolin 1 interaction, we conducted a comparative analysis of NSP4 in relation to two separate pools of proteins. The first pool included proteins collocated with the classical secretory pathway proteins, including caveolin 1, which traffick through the Golgi. The second pool included proteins collocated with a subset of caveolin 1, which traffick independently of the Golgi.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.