Clostridioides difficile (C. difficile) and human rotavirus (HRV) are leading causes of bacterial and viral gastroenteritis worldwide. Treatment and vaccination options for both pathogens have significant limitations. C. difficile infections are treated with antibiotics, which is paradoxical as C. difficile itself is associated with antibiotic usage. In the United States, two live oral attenuated vaccines (Rotarix and RotaTeq) are licensed for protection against HRV. Since receiving approval from the World Health Organization (WHO), Rotarix and RotaTeq have been widely implemented into global national childhood immunization schedules, with one report finding 59 countries using Rotarix and 25 using RotaTeq. However, these vaccines have much lower efficacy rates in low- and middle-income countries. Because of these caveats, there is an urgent need to generate novel prophylaxes and treatments for C. difficile and HRV. In order to address this need, animal models that replicate the nuances of each infection are imperative. We have developed gnotobiotic (Gn) pig models for each pathogen. Gn pigs infected with spores of the hypervirulent UK1 strain of C. difficile develop classical signs of infection, including watery diarrhea and weight loss. Gross necropsy reveals colonic distention and discoloration, and histopathological evaluation shows volcano lesions, pseudo membrane formation, and epithelial cell erosion. Gn pigs infected with a G4P[6] strain of HRV also display pathogen specific signs of infection, including diarrhea, fecal rotavirus shedding, and damaged intestinal villi. A dose response study of the G4P[6] strain revealed diarrhea and virus shedding occurred at all tested doses, however the most severe diarrhea and virus shedding, measured by cumulative diarrhea score, area under the curve (AUC) of diarrhea, peak virus titer, and AUC of virus shedding, were all detected in the highest dose group. Based on the presentation of clinical signs of infection, 105 fluorescent focus units was selected as the optimal challenge dose for future studies. These models enable us to test candidate therapeutics, but also elucidate unique replicative features of the pathogens. For example, we found that HRV can replicate in the salivary glands and nasal cavity of Gn pigs in addition to the small intestine. HRV infection primed immune responses in the ileum, tonsils, and facial lymph nodes; infection also induced high levels of systemic and mucosal rotavirus specific antibody responses. Moving forward, we hope to expand upon this replication study to identify what cell types within the glands are infected as well as look at local cellular immune responses to HRV infection. Additional future directions include determining the protective efficacy of next generation HRV vaccines and evaluating effectiveness of an engineered probiotic yeast in reducing severity of C. difficile infection and disease. The Gn pig models of C. difficile and G4P[6] HRV are clinically relevant, and they will continue to serve as useful tools to better our understanding of pathogenesis, infection, and prevention of these pathogens. / Doctor of Philosophy / Clostridioides difficile (C. difficile) and human rotavirus (HRV) both cause gastrointestinal related symptoms when they infect humans. Treatments available for C. difficile and HRV all have significant drawbacks. This represents a gap in knowledge which we aimed to fill by developing germ-free (gnotobiotic [Gn]) pig models of C. difficile and HRV infection and disease. Animal models that mimic the outcomes of disease seen in humans are essential for evaluating protectiveness of new therapeutics. The more similar the disease presentation, the more likely the treatment results will be translational to humans. We began with C. difficile; pigs were orally fed C. difficile and monitored for a week post infection for development of signs of infection. Inoculated pigs lost weight and developed diarrhea. Bacterial cells and toxins were isolated from fecal samples collected on various days post infection. Multiple changes were observed in infected pigs’ large intestinal tissues, including severe bleeding, tissue distension, and fluid buildup. Infected pigs also had significant upregulation of pro-inflammatory cytokines, indicating activation of the immune response. We performed a similar procedure for the establishment of the HRV model. Gn pigs were orally challenged with differing doses of G4P[6] HRV and followed for several days post infection. Consistent with HRV infection in children, the pigs developed watery diarrhea that lasted for multiple days. Small intestinal tissues collected at necropsy had several signs of damage, including blunted villi, fluid buildup, and immune cell invasion. These lesions were also consistent with HRV infection in humans. Taken all together, these results indicated successful establishment of both C. difficile and HRV models. While the primary goal of generating these models was to evaluate new treatments, a secondary goal was to use them to better our understanding of pathogen replication dynamics. For example, the small intestine was thought to be the primary site of HRV infection. Using a pig model of HRV, we expanded on this knowledge to show that HRV can replicate in the nose and salivary glands as well. Additionally, we found HRV infection to induce immune responses near the sites of infection, including the intestine, the tonsils, and the facial lymph nodes. Overall, these studies demonstrate the utility of germ-free pig models and are an important first step in generating more effective treatments for bacterial and viral infections.

As part of a larger quantitative microbial risk assessment (QMRA) study, the raw water intakes of 8 different drinking water treatment plants in Ontario were sampled for rotavirus. Group A rotavirus was detected and semi-quantified via RT-qPCR. Rotavirus was detected in 6 of 8 drinking water treatment plant raw water intakes at various sampling times during a 2 year period at estimated quantities of 0 – 513 viral genome copies/L water. As hypothesized, the virus counts showed a seasonal tendency with significant detection most likely to occur during the spring months and a correlation with turbidity measurements. To our knowledge this is the first study exploring the presence of rotavirus in Ontario source waters. With new proposed changes to the Health Canada guidelines regarding the viruses in drinking water, data on the presence of rotavirus in source waters is required for assessment of risk to public health. / Kingsclear First Nation

No description available.

With the absence of gut microbiota, gnotobiotic (Gn) pigs are a unique animal model for studying infection and immunity, and evaluating vaccine and therapeutics for human enteric pathogens. Here, we demonstrate Gn pigs as effective large animal models for human enteric viruses, through evaluating human enterovirus 71 (EV71) infection and immunity, and vaccine and therapeutics for human rotavirus (HRV). Gn pigs could be infected via oral or oronasal route, the natural route of infection. Infected pigs developed clinical signs including fever, neurological and respiratory signs, similar to those seen in human patients. Fecal shedding up to 18 days post infection and virus distribution in intestinal, respiratory and central nervous system tissues were observed. Strong mucosal and systemic T cell responses (IFN-γ producing CD4+ and CD8+ T cells) and systemic B cell responses (serum neutralizing antibodies) were also detected. The study demonstrates a novel large animal model for EV71 to investigate viral pathogenesis, immunity, and to evaluate vaccine and antiviral drugs. Using the well-established Gn pig model for HRV, the adjuvant and therapeutic effects of prebiotics rice bran (RB) and probiotics were evaluated. RB alone or RB plus probiotic Lactobacillus rhamnosus GG (LGG) and probiotic E. coli Nissle 1917 (EcN), were shown to protect against rotavirus diarrhea (80%-100% reduction in the incidence rate) significantly and display strong immune - stimulatory effects on the immunogenicity of an oral attenuated HRV (AttHRV) vaccine. Mechanisms for the adjuvant effect include stimulating the production of intestinal and systemic IFN-γ] producing T cells and promoting mucosal IgA antibody responses. The mechanisms for reducing rotavirus diarrhea include promoting LGG and EcN growth and colonization and host gut health, and maintaining gut integrity and permeability during rotavirus infection. We showed that RB plus LGG and EcN is a highly effective therapeutic regimen against HRV diarrhea. Together, these results indicated that Gn pigs may serve as an excellent animal model for the study of infection, immunity, vaccine and therapeutics for human enteric viruses. / Ph. D.

Norovirus and rotavirus are the most common viral causes of acute gastroenteritis among all age groups and in children under 5 years of age, respectively. Understanding the pathogenesis of the virus and correlates of protective immunity is fundamental to developing effective prevention and treatment strategies. Gnotobiotic (Gn) pigs are an attractive animal model for studying enteric viruses due to their similarities to humans, particularly in regards to the immune system and gastrointestinal anatomy and physiology. Here, to establish a reliable Gn pig model of human norovirus (HuNoV) infection and disease, we determined the median infectious dose (ID50) of a GII.4 2006b variant in pigs. We also evaluated the effects of age and administration of the cholesterol-lowering drug simvastatin on susceptibility to NoV infection. In neonatal pigs (4-5 days of age, the ID50 was determined to be 2.74 x 103 viral RNA copies. The ID50 was increased in 33-34 day old pigs (6.43 x 104), but decreased to <2.74 x 103 following simvastatin treatment in the same age group. Overall, the development of diarrhea, fecal virus shedding and small intestinal cytopathological changes confirmed the usefulness of the Gn pig as an appropriate animal model for studying HuNoVs. We also utilized the well-established Gn pig model of human rotavirus (HRV) infection and disease to evaluate adjunctive treatment options for HRV-induced diarrhea. We demonstrated that the anti-secretory drug racecadotril was capable of diminishing clinical signs of HRV infection and shortening duration of illness. Reduced dehydration in the racecadotril-treated pigs was evident by the significant gain in body weight compared to controls during the course of the study. We also determined that a high dose of the probiotic Lactobacillus acidophilus NCFM (LA) was able to reduce RV diarrhea severity and duration compared to a low dose. The difference in therapeutic potential was attributed to divergent effects in innate immunity pre- and post-challenge. High dose of LA (HiLA) induced an anti-inflammatory dendritic cell (DC) profile, characterized primarily by upregulation of TLR2 expression and production of cytokine IL-10. Conversely, low dose of LA (LoLA) upregulated TLR3 and TLR9 and increased secretion of cytokine IL-6. Additionally, HiLA induced both IFN-alpha and TNF-alpha responses in DCs, but LoLA was only able to increase the frequency of TNF-alpha-producing DCs. These results provide further support of Gn pigs as a highly applicable animal model for studying pathogenesis, innate immunity and therapeutics of human enteric viruses. / Ph. D. / Norovirus and rotavirus are the most common viral causes of acute gastroenteritis among all age groups and in children under 5 years of age, respectively. Understanding the pathogenesis of the virus and correlates of protective immunity is fundamental to developing effective prevention and treatment strategies. Gnotobiotic (Gn) pigs are an attractive animal model for studying enteric viruses due to their similarities to humans, particularly in regards to the immune system and gastrointestinal anatomy and physiology. Here, we established a reliable Gn pig model of human norovirus (HuNoV) infection and disease. Overall, the development of diarrhea, fecal virus shedding and small intestinal cytopathological changes confirmed the usefulness of the Gn pig as an appropriate animal model for studying HuNoVs. We also utilized the well-established Gn pig model of human rotavirus (HRV) infection and disease to evaluate adjunctive treatment options for HRV-induced diarrhea. We demonstrated that the anti-secretory drug racecadotril was capable of diminishing clinical signs of HRV infection and shortening duration of illness. We also determined that a high dose of the probiotic <i>Lactobacillus acidophilus</i> NCFM (LA) was able to reduce RV diarrhea severity and duration compared to a low dose. These results provide further support of Gn pigs as a highly applicable animal model for studying pathogenesis, innate immunity and therapeutics of human enteric viruses.

<p>In Molecular Medicine a wide range of methods are applied to analyze the genome to find genetic predictors of human disease. Apart from predisposing disease, genetic variations may also serve as genetic markers in the search for factors underlying complex diseases. Additionally, they provide a means to distinguish between species, analyze evolutionary relationships and subdivide species into strains. </p><p>The development and improvement of laboratory techniques and computational methods was a spin-off effect of the Human Genome Project. The same techniques for analyzing genomic sequence variations may be used independent of organism or source of DNA or RNA. In this thesis, methods for high-throughput analysis of sequence variations were developed, evaluated and applied. </p><p>The performance of several genotyping assays were investigated prior to genotyping 4000 samples in a co-operative genetic epidemiological study. Sequence variations in the estrogen receptor alpha gene were found to be associated with an increased risk of breast and endometrial cancer in Swedish women.</p><p>Whole genome amplification (WGA) enables large scale genetic analysis of sparse amounts of biobanked DNA samples. The performance of two WGA methods was evaluated using four-color minisequencing on tag-arrays. Our in-house developed assay and “array of arrays” format allow up to 80 samples to be analyzed in parallel on a single microscope slide. Multiple displacement amplification by the Φ29 DNA polymerase gave essentially identical genotyping results as genomic DNA. To facilitate accurate method comparisons, a cluster quality assessment approach was established and applied to assess the performance of four commercially available DNA polymerases in the tag-array minisequencing assay. </p><p>A microarray method for genotyping human group A rotavirus (HRV) was developed and applied to an epidemiological survey of infectious HRV strains in Nicaragua. The method combines specific capture of amplified viral sequences on microarrays with genotype-specific DNA-polymerase mediated extension of capture oligonucleotides with fluorescent dNTPs.</p>

In Molecular Medicine a wide range of methods are applied to analyze the genome to find genetic predictors of human disease. Apart from predisposing disease, genetic variations may also serve as genetic markers in the search for factors underlying complex diseases. Additionally, they provide a means to distinguish between species, analyze evolutionary relationships and subdivide species into strains. The development and improvement of laboratory techniques and computational methods was a spin-off effect of the Human Genome Project. The same techniques for analyzing genomic sequence variations may be used independent of organism or source of DNA or RNA. In this thesis, methods for high-throughput analysis of sequence variations were developed, evaluated and applied. The performance of several genotyping assays were investigated prior to genotyping 4000 samples in a co-operative genetic epidemiological study. Sequence variations in the estrogen receptor alpha gene were found to be associated with an increased risk of breast and endometrial cancer in Swedish women. Whole genome amplification (WGA) enables large scale genetic analysis of sparse amounts of biobanked DNA samples. The performance of two WGA methods was evaluated using four-color minisequencing on tag-arrays. Our in-house developed assay and “array of arrays” format allow up to 80 samples to be analyzed in parallel on a single microscope slide. Multiple displacement amplification by the Φ29 DNA polymerase gave essentially identical genotyping results as genomic DNA. To facilitate accurate method comparisons, a cluster quality assessment approach was established and applied to assess the performance of four commercially available DNA polymerases in the tag-array minisequencing assay. A microarray method for genotyping human group A rotavirus (HRV) was developed and applied to an epidemiological survey of infectious HRV strains in Nicaragua. The method combines specific capture of amplified viral sequences on microarrays with genotype-specific DNA-polymerase mediated extension of capture oligonucleotides with fluorescent dNTPs.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-13T12:18:47Z No. of bitstreams: 1 waldemir-de-castro-silveira.pdf: 669613 bytes, checksum: b8a4602ed033af1429638f26a5325f0d (MD5) / Made available in DSpace on 2012-11-13T12:18:47Z (GMT). No. of bitstreams: 1 waldemir-de-castro-silveira.pdf: 669613 bytes, checksum: b8a4602ed033af1429638f26a5325f0d (MD5) Previous issue date: 2005 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O rotavírus humano (RV) pode ser detectado prontamente pelo método de aglutinação do látex (LTE), com elevada sensibilidade e especificidade, através da reação cruzada com o anticorpo anti-rotavírus decarneiro. As partículas de látex revestidas com a imunoglobulina G anti-rotavírus, são especificamente aglutinadas na presença do RV, com resultados macroscopicamente evidentes dentro de poucos minutos. Para a sensibilidade e a especificidade do método de LTE proposto, comparou-se com outro método similar (Rota-Kit Slidex®; BioMérieuxl), um total de 81 amostras fecais de crianças até 5 anos com gastroenterite aguda. Com 36 amostras diarréicas RV-positivos e 45 RV-negativos, a ensibilidade analítica do teste de LTE proposto foi > 99,99%, e a especificidade analítica também foi > 99,99%. O LTE demonstrou ser capaz de detectar partículas de rotavírus em fezes diarréicas em concentrações 20 vezes menores que o limite de detecção do Rota-Kit Slidex®. A freqüência de testes positivos de LTE pareceu ser proporcionalà concentração de viríons na amostra fecal. O LTE demonstrou uma sensibilidade de 94,0% (34 de 36 positivos), e uma especificidade > 99,99% (45de 45 negativas) quando comparado com o Rotazyme II®, um ensaio imunoenzimático (Abbott Laboratories). O método do LTE é rápido, facilmenteutilizável para detectar o RV em amostra fecal de crianças e é apropriado para a aplicação como um teste diagnóstico simples, principalmente para triagem de grandes grupos de pacientes. O LTE é estável por pelo menos 2 anos searmazenado sob refrigeração (4°C) e se mostra útil para laboratórios de pequeno porte, para rotinas ambulatoriais, de emergências e de triagem e finalmente, para discriminar amostras negativas que poderão então ser testadas por testes mais sensíveis. / Human rotavírus (HRV) can readily be detected by the latex agglutination assay method (LTE), with raised sensitivity and specificity, based on crossed reaction with the anti-rotavirus sheep antibody. The latex particles coated with anti-rotavirus immunoglobulin G, agglutinates specifically when in contact with HRV in few minutes, with evident macroscopically results. The sensitivity and the specificity performance of the proposed method (LTE), was compared with another similar method (Rota-Kit Slidex®, BioMérieux), a total of 81 specimens from children with acute gastroenteritis were tested. With 36 HRV positive samples and 45 HRV negative samples, the analytical sensitivity of LTE was better than 99,99% and the analytical specificity, also, was better than 99,99%. The LTE demonstrated to be capable to detect particles of HRV in fecal samples in concentrations20 lesser times that the limit of detection of Rota-Kit Slidex®. The frequency of positive tests of LTE seemed to be proportional to the concentration of virions in the fecal sample. The LTE demonstrated a sensitivity of 94,0%(34 of 36 positives), and a specificity >99,99% (45 of 45 negatives) whencompared with Rotazyme II® (Abbott Laboratories). The method of the LTE is fast and easily usable to detect HRV in children fecal sample and is appropriate for application as a test of simple diagnosis, mainly for screening of great groups of patients. The LTE is stable per at least 2 yearsif stored under refrigeration (4°C) and it shows useful for small laboratories, clinic routines, emergence rooms and screenings and finally to discriminate negative samples that could then be tested by more sensible tests.

Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012