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Objectifs. La vaccination des nouveau-nés contre le rotavirus est recommandée par l'OMS dans les pays où un impact significatif de santé publique est à prévoir, chaque pays doit établir le fardeau des maladies liées au rotavirus et établir le coût-bénéfice potentiel de l'implantation de la vaccination. Le fardeau du rotavirus n'est pas encore bien déterminé au Canada et au Québec, les données épidémiologiques disponibles incluent rarement l'impact sur les unités d'observation pédiatrique de courte durée, les urgences et les visites externes de façon simultanée. L'objectif principal de cette étude est de déterminer le fardeau de la gastro-entérite aiguë liée au rotavirus (GEAR) sur l'ensemble du système de santé de la région de l'Estrie. Méthode. Il s'agit d'une étude de cohorte rétrospective qui incluait les enfants < 5 ans consultant en Estrie pour gastro-entérite aiguë (GEA) selon les codes CIM-9/10 correspondants de novembre 2002 à octobre 2008. Les données d'hospitalisations et de l'urgence provenaient de l'entrepôt de données cliniques CIRESSS du CHUS, celles des consultations externes de la RAMQ. La recherche de rotavirus n'étant pas systématique, nous avons utilisé deux méthodes d'estimation indirecte reconnues (Winter Residual Estimation et méthodes des coefficients de Brandt) pour extrapoler les taux de GEAR. Résultats. Sur les 6 années d'étude, ont été recensées 1435 hospitalisations (hospitalisations standard : 598, hospitalisations de moins de 24h : 837), 3631 consultations à l'urgence et 6220 consultations externes pour GEA. La part liée au rotavirus selon les méthodes de Brandt et WRE était respectivement de 404 à 666 hospitalisations, 1009 à 1361 consultations à l'urgence et 1613 à 1687 consultations ambulatoires. Pour la population des enfants de moins de 5 ans en Estrie les taux d'incidence annuels d'hospitalisations pour GEAR étaient estimés entre 45 et 74/10000, pour les passages à l'urgence entre 113 à 152/10000 et pour les consultations externes entre 180 à 188/10000. La courbe épidémique objectivait un pic annuel survenant durant les mois de mars et d'avril. La majorité d'enfants hospitalisés pour GEAR avaient moins de 2 ans (65%). La durée médiane d'hospitalisation en unité standard était de 53 heures et plus de 90% des enfants hospitalisés ont bénéficié d'une réhydratation par voie intraveineuse. Nous avons identifié 83 GEA nosocomiales. Il n'y a eu aucun décès lié à la GEA pendant l'étude. Conclusion. La plupart des études rétrospectives disponibles sous-estiment le poids de la maladie en omettant les hospitalisations de courte durée. De plus, il s'agit de la première étude au Canada évaluant de façon globale le fardeau de la maladie liée au rotavirus sur l'ensemble du système de santé. Les résultats de cette étude pourront servir à l'évaluation de l'intégration du vaccin anti rotavirus au programme d'immunisation du Québec.

published_or_final_version / Community Medicine / Master / Master of Public Health

Rotavírus têm sido identificados mundialmente como o mais importante agente etiológico de diarréias agudas não-bacterianas em animais jovens de várias espécies, incluindo a humana. Foi desenvolvido e avaliado um método de ELISA tipo “duplo-sanduíche" para a detecção de rotavírus a partir de material fecal. Para tanto, a amostra NCDV de rotavírus do grupo A foi propagada em cultivo celular com células MA-104. O vírus foi concentrado por ultracentrifugação e inoculado em coelhos e carneiros. Em seguida, as frações IgG, oriundas de amostras de soro dos animais, foram purificadas por cromatografia de troca iônica e absorvidas com soro total de ambas espécies animais, utilizando-se polímero de glutaraldeído, de modo a eliminar reações inespecíficas. A presença do rotavírus foi detectada pela IgG de carneiros e revelada pela IgG de coelho, usando como conjugado IgG de cabra anti-IgG de coelho conjugada à peroxidase. Os valores de diluição dos componentes do ELISA e o valor do ponto-de-corte foram definidos usando-se 26 amostras fecais (13 positivas e 13 negativas) de leitões, tendo como prova padrão a eletroforese em gel de poliacrilamida (PAGE). Aplicado a um painel constituído de 86 amostras fecais diarréicas de leitões, os resultados do ELISA foram: 100% de sensibilidade; 98,79% de especificidade, com uma concordância de 98,83%. A variância entre 86 repetições da mesma amostra foram 0,001 (para a amostra positiva) e 0,0002 (para a amostra negativa). Estes resultados demonstram que este ELISA é um teste sensível e específico para o diagnóstico de rotavírus a partir de material fecal. / Rotaviruses have been identified worldwide as a major etiologic agent of acute nonbacterial diarrhea in the young of many species, including humans. In this investigation was developed and evaluated a “double-sandwich" antibody ELISA method for detection of rotavirus from stool specimens. For that, the NCDV strain of rotavirus group A was serially cultivated in MA-104 cell culture. The virus was concentrated by ultra-centrifugation and inoculated in rabbits and sheeps. After that, the IgG of serum samples of the animals was purified by ion-exchange chromatography and absorbed with whole serum of both animal species using a glutaraldehyde polymer, in order to eliminate inespecific reactions. The presence of rotavirus was detected by the sheep’s IgG and revelated by the rabbit’s IgG, using a anti-rabbit IgG peroxidase conjugate developed in goat. The values of diluition of the components of the ELISA and the cut-off value were defined using 26 fecal samples (13 positive and 13 negative) of piglets. Following this procedure, the test was employed in a panel of 86 fecal samples from piglets with diarrhea, using as standard the polyacrilamide gel electrophoresis (PAGE) test. The results of the ELISA were: 100% of sensivity; 98.79% of specificity, with an agreement of 98.83%. The variance between 86 repetitions of the same sample were 0.001 (for one positive sample) and 0.0002 (for one negative sample). These results showed that this ELISA is an sensitive and specific screening test for rotavirus diagnosis from fecal material.

by Graham Neil Thomas. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 167-191). / Abstract --- p.i / Contents --- p.iii / List of tables --- p.viii / List of figures --- p.x / Abbreviations --- p.xi / Acknowledgements --- p.xiii / Chapter Chapter 1 - --- Introduction / Chapter 1.1 --- Introduction to the genus rotavirus --- p.1 / Chapter 1.2 --- General characteristics of rotavirus --- p.3 / Chapter 1.3 --- Clinical and epidemiological characteristics of rotaviral infections --- p.4 / Chapter 1.3.1 --- Clinical features of rotavirus infection --- p.4 / Chapter 1.3.2 --- Nosocomial rotavirus infection --- p.4 / Chapter 1.3.3 --- Morbidity and Mortality of rotavirus diarrhoea --- p.5 / Chapter 1.3.3.1 --- Seasonal distribution of rotavirus in temperate regions … --- p.5 / Chapter 1.3.3.2 --- Rotavirus infections in developing countries --- p.6 / Chapter 1.3.3.3 --- Rotavirus infections in developed countries --- p.6 / Chapter 1.3.4 --- Host Resistance to rotavirus infection --- p.7 / Chapter 1.3.5 --- Pathogenesis --- p.9 / Chapter 1.4 --- Vaccine development in rotavirus prevention --- p.10 / Chapter 1.4.1 --- Attenuated HRV as candidate vaccine strains --- p.11 / Chapter 1.4.2 --- Animal RV candidate vaccine strains (Jennerian approach)..… --- p.11 / Chapter 1.4.3 --- Intra- and interspecies reassortants vaccine strains --- p.12 / Chapter 1.4.4 --- Passive immunisation --- p.13 / Chapter 1.5 --- Laboratory diagnosis of rotavirus infections --- p.14 / Chapter 1.5.1 --- Detection of rotavirus --- p.14 / Chapter 1.5.2 --- Negative stain electron microscopy (EM) --- p.15 / Chapter 1.5.3 --- Immunological assays for the detection of rotavirus antigens --- p.15 / Chapter 1.5.4 --- Polyacrylamide gel electrophoresis (PAGE) of RV RNA --- p.16 / Chapter 1.5.5 --- Nucleic acid probe hybridisation assays --- p.17 / Chapter 1.6 --- Antigenic classification of rotaviruses --- p.17 / Chapter 1.6.1 --- Rotavirus groups --- p.17 / Chapter 1.6.2 --- Rotavirus subgroups --- p.18 / Chapter 1.6.3 --- Rotavirus serotypes --- p.19 / Chapter 1.6.4 --- Rotavirus genogroups --- p.21 / Chapter 1.7 --- Molecular biology of rotavirus --- p.21 / Chapter 1.7.1 --- Rotavirus genomic organisation --- p.21 / Chapter 1.7.2 --- Gene coding assignments --- p.22 / Chapter 1.7.3 --- Genome and protein structure of rotavirus VP7 --- p.22 / Chapter 1.8 --- Reverse transcriptase-Polymerase chain reaction (RT-PCR) for the genotyping of rotavirus --- p.32 / Chapter 1.8.1 --- Prevention of contamination in RT-PCR --- p.34 / Chapter 1.9 --- Objectives of the study --- p.36 / Chapter Chapter 2 - --- Methods / Chapter 2.1 --- Collection of specimens --- p.38 / Chapter 2.2 --- Standard rotavirus strains --- p.38 / Chapter 2.3 --- Tissue culture techniques --- p.39 / Chapter 2.3.1 --- Growth of MA104 cell line --- p.39 / Chapter 2.3.2 --- Subculturing of MA104 cell line --- p.40 / Chapter 2.3.3 --- Virus propagation and isolation --- p.40 / Chapter 2.3.4 --- Harvesting and purification of viral particles --- p.41 / Chapter 2.4 --- Rotavirus electropherotyping by PAGE --- p.42 / Chapter 2.4.1 --- RNA extraction --- p.42 / Chapter 2.4.2 --- Polyacrylamide gel electrophoresis (PAGE) --- p.43 / Chapter 2.4.3 --- Silver staining of RNA in polyacrylamide gels --- p.43 / Chapter 2.5 --- Enzyme immunoassays in rotavirus typing --- p.44 / Chapter 2.5.1 --- Preparation of monoclonal antibodies (mAb) from hybridoma cell lines --- p.44 / Chapter 2.5.1.1 --- Growth of hybridoma cell lines --- p.44 / Chapter 2.5.1.2 --- Preparation of the ascitic fluid - monoclonal Abs --- p.45 / Chapter 2.5.2 --- Confirmation of mAb activity by immunofluorescence (IF)..… --- p.46 / Chapter 2.5.2.1 --- Preparation of virus-infected cells --- p.46 / Chapter 2.5.2.2 --- Confirmation of the serotype specificity of the mAb by immunofluorescence microscopy --- p.47 / Chapter 2.5.3 --- Polyclonal hyperimmune antisera against rotavirus --- p.48 / Chapter 2.5.4 --- Immunoglobulin purification --- p.48 / Chapter 2.5.5 --- Monoclonal antibody-based serotyping and subgrouping EIA --- p.49 / Chapter 2.6 --- Reverse transcription-Polymerase Chain Reaction genotyping of rotavirus (RT-PCR) --- p.53 / Chapter 2.6.1 --- Primers used in RT-PCR --- p.53 / Chapter 2.6.1.1 --- Preparation of oligonucleotide primers for RV genotyping --- p.53 / Chapter 2.6.1.2 --- Detachment of the oligonucleotide from the column --- p.54 / Chapter 2.6.1.3 --- Purification of the oligonucleotides --- p.55 / Chapter 2.6.1.4 --- Confirmation of oligonucleotide synthesis --- p.58 / Chapter 2.6.2 --- Preparation of specimens --- p.59 / Chapter 2.6.3 --- Reverse transcription of genomic RNA template and PCR…… --- p.64 / Chapter 2.6.4 --- PCR genotyping using full-length cDNA template --- p.64 / Chapter 2.6.5 --- Product identification --- p.65 / Chapter Chapter 3 - --- Results / Chapter 3.1 --- Epidemiology of rotavirus infections in Hong Kong --- p.70 / Chapter 3.2 --- RT-PCR genotyping of rotavirus --- p.74 / Chapter 3.3 --- Seasonal distribution of rotavirus genotypes --- p.76 / Chapter 3.4 --- Comparison of RT-PCR genotyping of the VP7 gene with mEIA…… --- p.79 / Chapter 3.5 --- Relationship between electropherotyping and genotyping by RT-PCR --- p.91 / Chapter 3.6 --- Atypical rotavirus strains --- p.92 / Chapter 3.7 --- Specimens exhibiting multiple genotype specificities --- p.93 / Chapter 3.8 --- HRV RT-PCR genotype primers --- p.95 / Chapter Chapter 4 - --- Discussion / Chapter 4.1 --- Epidemiology of rotavirus infections in Hong Kong --- p.101 / Chapter 4.2 --- RT-PCR genotyping of rotavirus --- p.103 / Chapter 4.2.1 --- Modifications to methodology --- p.104 / Chapter 4.2.2 --- Rotavirus genotypes --- p.107 / Chapter 4.3 --- Comparison of RT-PCR genotyping with mEIA typing --- p.111 / Chapter 4.4 --- Relationship between electropherotyping and RT-PCR genotyping --- p.113 / Chapter 4.5. --- Rotavirus genotype distribution in Hong Kong --- p.115 / Chapter 4.6 --- Specimens containing atypical rotavirus strains --- p.119 / Chapter 4.7 --- Stool specimens exhibiting multiple rotavirus genotypes identified by RT-PCR --- p.122 / Chapter 4.8 --- Specificity analysis of RT-PCR primers --- p.124 / Chapter 4.8.1 --- 5non-coding region (1-28) - primer BEG9 --- p.125 / Chapter 4.8.2 --- 3non-coding region (1033/6-1062) - primers END9/RVG9. --- p.125 / Chapter 4.8.3 --- Variable region A (165-198) - primer aAT8 (G8) --- p.125 / Chapter 4.8.4 --- Variable region B (309-351) - primer aBT1 (G1) --- p.126 / Chapter 4.8.5 --- Variable region C (408-438) - primer aCT2 (G2) --- p.127 / Chapter 4.8.6 --- Variable region D (477-504) - primer aDT4 (G4) --- p.127 / Chapter 4.8.7 --- Variable region E (672-711) - primer aET3 (G3) --- p.128 / Chapter 4.8.8 --- Variable region F (747-776) - primer aFT9 (G9) --- p.128 / Chapter 4.9 --- Future developments of the study --- p.131 / Appendices / Chapter A1 --- Materials --- p.135 / Chapter A2.1-2.9 --- Distribution of electropherotypes between July 1985 and April 1994 --- p.145 / Chapter A3.1-3.11 --- Individual rotavirus strain electropherotypes --- p.153 / Chapter A4.1-4.8 --- "Comparison of nucleotide sequences for the 3', 5'-non-coding regions and variable regions A to F for the VP7 gene" --- p.158 / Chapter A4.9 --- Rotavirus strains and nucleotide sequence references for the VP7 gene --- p.166 / References

Rotavírus têm sido identificados mundialmente como o mais importante agente etiológico de diarréias agudas não-bacterianas em animais jovens de várias espécies, incluindo a humana. Foi desenvolvido e avaliado um método de ELISA tipo “duplo-sanduíche” para a detecção de rotavírus a partir de material fecal. Para tanto, a amostra NCDV de rotavírus do grupo A foi propagada em cultivo celular com células MA-104. O vírus foi concentrado por ultracentrifugação e inoculado em coelhos e carneiros. Em seguida, as frações IgG, oriundas de amostras de soro dos animais, foram purificadas por cromatografia de troca iônica e absorvidas com soro total de ambas espécies animais, utilizando-se polímero de glutaraldeído, de modo a eliminar reações inespecíficas. A presença do rotavírus foi detectada pela IgG de carneiros e revelada pela IgG de coelho, usando como conjugado IgG de cabra anti-IgG de coelho conjugada à peroxidase. Os valores de diluição dos componentes do ELISA e o valor do ponto-de-corte foram definidos usando-se 26 amostras fecais (13 positivas e 13 negativas) de leitões, tendo como prova padrão a eletroforese em gel de poliacrilamida (PAGE). Aplicado a um painel constituído de 86 amostras fecais diarréicas de leitões, os resultados do ELISA foram: 100% de sensibilidade; 98,79% de especificidade, com uma concordância de 98,83%. A variância entre 86 repetições da mesma amostra foram 0,001 (para a amostra positiva) e 0,0002 (para a amostra negativa). Estes resultados demonstram que este ELISA é um teste sensível e específico para o diagnóstico de rotavírus a partir de material fecal. / Rotaviruses have been identified worldwide as a major etiologic agent of acute nonbacterial diarrhea in the young of many species, including humans. In this investigation was developed and evaluated a “double-sandwich” antibody ELISA method for detection of rotavirus from stool specimens. For that, the NCDV strain of rotavirus group A was serially cultivated in MA-104 cell culture. The virus was concentrated by ultra-centrifugation and inoculated in rabbits and sheeps. After that, the IgG of serum samples of the animals was purified by ion-exchange chromatography and absorbed with whole serum of both animal species using a glutaraldehyde polymer, in order to eliminate inespecific reactions. The presence of rotavirus was detected by the sheep’s IgG and revelated by the rabbit’s IgG, using a anti-rabbit IgG peroxidase conjugate developed in goat. The values of diluition of the components of the ELISA and the cut-off value were defined using 26 fecal samples (13 positive and 13 negative) of piglets. Following this procedure, the test was employed in a panel of 86 fecal samples from piglets with diarrhea, using as standard the polyacrilamide gel electrophoresis (PAGE) test. The results of the ELISA were: 100% of sensivity; 98.79% of specificity, with an agreement of 98.83%. The variance between 86 repetitions of the same sample were 0.001 (for one positive sample) and 0.0002 (for one negative sample). These results showed that this ELISA is an sensitive and specific screening test for rotavirus diagnosis from fecal material.

Rotavirus (RV) nonstructural protein 4 (NSP4) is a multifunctional glycoprotein that induces secretory diarrhea in mouse pups in the absence of other viral proteins. The intracellular transport route(s) and functional mechanism(s) of NSP4 are poorly understood; however, the recent association of the enterotoxin with cellular caveolin-1 may provide a link between NSP4 transport and function. To determine if NSP4 traffics to a specific subset of lipid rafts at the plasma membrane (PM), we isolated caveolae from a PM-enriched fraction with a new method that yielded endoplasmic reticulum (ER)-free caveolae membranes with a unique membrane structure and composition. Comparison of these caveolae with other detergent- and non-detergent-extracted membranes revealed that each caveolae/raft fraction contained caveolae markers; however, only our PM caveolae fraction mimicked the membrane structure and sterol exchange dynamics of intact PM without ER or non-raft PM contaminants. When these PM caveolae were isolated from RV-infected cells, full-length, high-mannose glycosylated NSP4 was present. Confocal imaging showed association of NSP4 with caveolin-1 moving from perinuclear and cytoplasmic sites toward the PM as the infection progressed. Fluorescent imaging also indicated exposure of the NSP4 Cterminus at the exofacial PM surface without transport of the enterotoxin through the Golgi apparatus. Surface-specific biotinylation was used to confirm NSP4 exposure at the surface of infected MDCK cells and to determine that the exposed protein was fulllength and high-mannose glycosylated. This study presents an ER contaminant-free PM caveolae isolation methodology, identifies the presence of full-length, high-mannose glycosylated NSP4 in both PM caveolae and exposed at the cell surface, and confirms the Golgi-bypassing nature of NSP4 ER to PM transport in RV-infected MDCK cells.

Rotavirus NSP4, the first described viral enterotoxin, localizes to the plasma membrane of infected cells, possibly through interaction with caveolin-1. A direct interaction between NSP4 and caveolin-1, the structural protein of caveolae, was shown by yeast two-hybrid, peptide binding, and FRET analyses. To dissect the precise NSP4 binding domain to caveolin-1, mutants were prepared by altering either the charged or hydrophobic face of the NSP4 C-terminal amphipathic alpha-helix and examined for binding to caveolin-1. Replacing six charged residues with alanine (FLNSP4Ala) disrupted the charged face, while the hydrophobic face was disrupted by replacing selected hydrophobic residues with charged amino acids (aa) (FLNSP4HydroMut). In yeast two-hybrid and peptide binding assays, FLNSP4Ala retained its binding capacity, whereas FLNSP4HydroMut failed to bind caveolin-1. Mutants were generated with an Nterminal truncated clone (NSP446-175), which removed the hydrophobic domains and aided in yeast-two hybrid assays. These mutants exhibited the same binding pattern as FLNSP4 confirming that the N-terminus of NSP4 lacks the caveolin-1 binding site and NSP446-175 is sufficient for binding. Seven additional mutants were prepared from NSP4HydroMut in which individually charged residues were reverted to the original hydrophobic aa or were replaced with alanine. Analyses of the interaction of these revertants with caveolin-1 localized the NSP4 binding domain to one critical hydrophobic aa (L116) and one or two additional aa (I113, L127, and/or L134) on the hydrophobic face. Those mutants that bound caveolin-1 bound both the N- and C-terminal caveolin-1 peptides, but lacked binding to a centrally located peptide. These data suggest conformational and hydrophobic constraints play a role in the NSP4-caveolin-1 association. The mutant NSP4 molecules also were evaluated for transport to the plasma membrane. Mammalian cells were transfected with FLNSP4, FLNSP41-175Ala, and NSP41-175HydroMut plasmid DNA, surface biotinylated, and examined by IFA or Western blot for NSP4 expression. Epifluorescence revealed FLNSP4 and FLNSP4Ala were exposed on the cell surface in the absence of other viral proteins, whereas NSP4HydroMut remained intracellular. Further, NSP4-transfected cells displayed an intracellular association of with caveolin-1 or the caveolin-1 chaperone complex proteins. These data indicate NSP4 interacts with caveolin-1 in the absence of other viral proteins.

Rotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular calcium through multiple mechanisms that promote abnormal ion transport and subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially interact with model membranes mimicking caveolae in lipid composition and radius of curvature. Our laboratory has recently reported the colocalization and coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae. Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least one terminus were capable of binding the NSP4 bait. An in vitro binding assay confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1 residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates NSP4 signaling and/or intracellular trafficking.