Genotyping of the rotavirus VP7 gene by the reverse transcription-polymerase chain reaction.

Chinese University of Hong Kong 1995

by Graham Neil Thomas.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1995.
Includes bibliographical references (leaves 167-191).
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
http://library.cuhk.edu.hk/record=b5888528
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Rotavirus--Genetics; Polymerase chain reaction; Genotype; Text; bibliography

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xiii, 191 leaves : ill. (some mounted col.) ; 30 cm.
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959213879

CHINESE UNIV OF HONG KONG
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