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Louis, Tong Kwok-leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 121-130). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.III / LIST OF CONTENTS --- p.IV / LIST OF TABLES --- p.VIII / LIST OF FIGURES --- p.X / ABBREVIATIONS --- p.XII / GENERAL ABBREVIATIONS --- p.XII / VARIOUS NOMENCLATURES OR ABBREVIATIONS FOR SRSVS --- p.XIII / OBJECTIVES OF THE STUDY --- p.XIV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- HISTORICAL PERSPECTIVE --- p.2 / Chapter 1.2 --- CLINICAL FEATURES OF HUMAN SRSV INFECTION --- p.10 / Chapter 1.3 --- EPIDEMIOLOGY --- p.12 / Chapter 1.4 --- PHYSICAL CHARACTERISTICS OF SRSV --- p.14 / Chapter 1.5 --- STABILITY OF NORWALK VIRUS --- p.15 / Chapter 1.6 --- DIAGNOSTIC TOOLS FOR SRSVS --- p.15 / Chapter 1.7 --- GENOMIC ORGANIZATION OF SRSVS --- p.16 / Chapter 1.8 --- MOLECULAR TECHNOLOGIES --- p.19 / Chapter CHAPTER 2 --- MATERIALS --- p.20 / Chapter 2.1 --- FAECAL SAMPLES FROM 1986 TO 1992 --- p.21 / Chapter 2.2 --- OTHER FAECAL SAMPLES FROM 1995 TO 1998 --- p.21 / Chapter 2.3 --- AGE GROUPS OF ALL THE SAMPLES --- p.21 / Chapter CHAPTER 3 --- METHODS --- p.23 / Chapter 3.1 --- EXTRACTION OF RNA FROM PATIENT STOOL OR VOMIT SAMPLES --- p.24 / Chapter 3.2 --- REVERSE TRANSCRIPTION - POLYMERASE CHAIN REACTION (RT-PCR) FOR SRSVS --- p.25 / Chapter 3.2.1 --- Principle of RT-PCR assay for SRSV --- p.25 / Chapter 3.2.2 --- Reverse transcription (cDNA Synthesis) --- p.26 / Chapter 3.2.3 --- Polymerase chain reaction --- p.26 / Chapter 3.2.4 --- Electrophoresis (in the PCR product room) --- p.31 / Chapter 3.2.5 --- Controls for PCR assay --- p.32 / Chapter 3.2.6 --- Interpretation of SRSV polymerase gene PCR --- p.32 / Chapter 3.3 --- RT-PCR USING INOSINE CONTAINING PRIMERS FOR THE CAPSID REGIONS --- p.34 / Chapter 3.3.1 --- RT-PCR for the capsid region of SRSV genome --- p.34 / Chapter 3.3.2 --- Interpretation of SRSV capsid gene PCR --- p.36 / Chapter 3.4 --- SOLID PHASE IMMUNE ELECTRON MICROSCOPY FOR THE DETECTION OF SRSV --- p.37 / Chapter 3.5 --- OPTIMIZATION OF CONDITIONS FOR SRSV RT-PCR --- p.37 / Chapter 3.5.1 --- Titration of primers --- p.37 / Chapter 3.5.2 --- Titration of MgCl2 --- p.38 / Chapter 3.5.3 --- "Titration ofdNTPs, MgCl2 and Taq polymerase (pH 9.0)" --- p.38 / Chapter 3.6 --- SPECIFICITY OF SRSV RT-PCR --- p.38 / Chapter 3.7 --- PURIFICATION OF PCR PRODUCTS PRIOR TO CLONING …… --- p.38 / Chapter 3.8 --- CLONING OF THE PURIFIED DNA INTO pGEM-T EASY VECTOR --- p.39 / Chapter 3.8.1 --- Introduction --- p.39 / Chapter 3.8.2 --- Sequence of the pGEM-T Easy Vector --- p.42 / Chapter 3.8.3 --- Ligation --- p.44 / Chapter 3.8.4 --- Transformation of competent bacterial cells --- p.44 / Chapter 3.8.5 --- Small-scale preparations of plasmid DNA --- p.45 / Chapter 3.8.6 --- Purification of miniprep using QIAprep Miniprep --- p.45 / Chapter 3.8.7 --- Restriction analysis of small-scale preparations of plasmid DNA --- p.45 / Chapter 3.9 --- CYCLE SEQUENCING OF CLONED SRSV AMPLICONS --- p.46 / Chapter 3.9.1 --- Targets for Sequencing --- p.46 / Chapter 3.9.2 --- Procedures of cycle sequencing --- p.46 / Chapter 3.9.3 --- Gel electrophoresis --- p.48 / Chapter 3.9.4 --- Sequencing conditions --- p.49 / Chapter 3.10 --- SEQUENCE ANALYSIS --- p.49 / Chapter CHAPTER 4 --- REAGENTS AND BUFFERS --- p.51 / Chapter 4.1 --- REAGENTS AND BUFFERS FOR RNA EXTRACTION --- p.52 / Chapter 4.2 --- REAGENTS AND BUFFERS FOR REVERSE TRANSCRIPTION (cDNA SYNTHESIS) --- p.52 / Chapter 4.3 --- REAGENTS AND BUFFERS FOR PCR --- p.53 / Chapter 4.4 --- GEL ELECTROPHORESIS OF PCR PRODUCTS --- p.53 / Chapter 4.5 --- PURIFICATION OF PCR PRODUCTS --- p.54 / Chapter 4.6 --- REAGENTS FOR CLONING THE DNA INSERT INTO pGEM-T EASY VECTOR --- p.54 / Chapter 4.6.1 --- "pGEM-T Easy Vector System (Promega Corporation, USA)" --- p.54 / Chapter 4.6.2 --- Isopropylthio-β-D-galactoside (IPTG) stock solution --- p.54 / Chapter 4.6.3 --- X-Gal --- p.54 / Chapter 4.6.4 --- Luria-Bertani (LB) medium --- p.55 / Chapter 4.6.5 --- LB plates with ampicillin --- p.55 / Chapter 4.6.6 --- LB plates with ampicillin/IPTG/X-Gal --- p.55 / Chapter 4.6.7 --- SOC medium --- p.55 / Chapter 4.6.8 --- Mini-prep purification --- p.56 / Chapter 4.6.9 --- Mini-prep analysis --- p.56 / Chapter 4.6.9.1 --- Lambda DNA-Hind IIIφX-174 DNA-Hae III Digest --- p.56 / Chapter 4.6.9.2 --- Not I --- p.58 / Chapter 4.7 --- REAGENTS AND BUFFERS FOR CYCLE SEQUENCING --- p.58 / Chapter 4.7.1 --- SequiTherm EXCEĹёØ II Long-Rea´dёØ DNA Sequencing Kit-AL´FёØ --- p.58 / Chapter 4.7.2 --- Sequencing primers --- p.59 / Chapter 4.8 --- REAGENTS FOR SEQUENCING GEL CASTING --- p.59 / Chapter CHAPTER 5 --- RESULTS --- p.61 / Chapter 5.1 --- RESULTS OF RT-PCR OPTIMIZATION --- p.62 / Chapter 5.1.1 --- Magnesium chloride and pH of PCR reaction buffer --- p.62 / Chapter 5.1.2 --- Concentration of primers --- p.64 / Chapter 5.1.3 --- "Titration of dNTPs, MgCl2 and Taq polymerase (pH 9.0)" --- p.65 / Chapter 5.2 --- RESULT OF SENSITIVITY TEST --- p.66 / Chapter 5.3 --- RESULTS OF SPECIFICITY TEST --- p.67 / Chapter 5.4 --- RESULTS OF THE PCR USING INOSINE CONTAINING POL PRIMERS --- p.70 / Chapter 5.5 --- RESULTS OF PCR USING INOSINE CONTAINING CAPSID PRIMERS --- p.73 / Chapter 5.6 --- RESULTS OF SOME SAMPLES RETESTED BY SPIEM --- p.75 / Chapter 5.7 --- RESULTS OF SPORADIC OUTBREAKS --- p.77 / Chapter 5.7.1 --- A sporadic outbreak in 1996 --- p.77 / Chapter 5.7.2 --- Sporadic outbreak in a kindergarten in 1997 --- p.79 / Chapter 5.7.3 --- Sporadic outbreak at a hotel in 1998 --- p.79 / Chapter 5.7.4 --- Application of the RT-PCR to contaminated shellfish --- p.80 / Chapter 5.8 --- RESULTS OF MINI PREP ANALYSIS WITH NOT I DIGESTION --- p.85 / Chapter 5.9 --- RESULT OF ELECTROPHEROGRAM OF A SELECTED SPECIMEN FROM THE AUTOMATIC SEQUENCING --- p.86 / Chapter 5.9 --- RESULT OF ELECTROPHEROGRAM OF A SELECTED SPECIMEN FROM THE AUTOMATIC SEQUENCING --- p.86 / Chapter 5.10 --- RESULTS OF ALL TRIMMED DNA SEQUENCES --- p.87 / Chapter CHAPTER 6 --- DISCUSSION --- p.112 / REFERENCES --- p.122 / APPENDIX --- p.131 / APPENDIX I: Electron micrograph of SRSV particles --- p.132 / APPENDIX II: Confirmation for specificity test --- p.133 / APPENDIX III: Sequencing amplicons using capsid primers --- p.135 / APPENDIX IV: Sequencing amplicons (outbreak) using pol primers --- p.136 / APPENDIX V: Electropherogram (direct sequencing) --- p.138 / APPENDIX VI: Other RT-PCR results using pol primers --- p.139 / APPENDIX VII: Results of RT-PCR using capsid primers --- p.149 / APPENDIX VIII: Mini prep analysis --- p.158

Vírus causadores de gastroenterite, descritos como pequenos vírus de estrutura arredondada (Small Round Structured Viruses-SRSV), foram detectados em extratos de ostras Crassostrea spp., coletadas em regiões distintas do litoral do Estado de São Paulo, utilizando a Reação em Cadeia por Polimerase com transcrição reversa (RT-PCR).Treze lotes de amostras, contendo 10 g de estômagos e divertículos, foram processados para extração e concentração das partículas virais, mediante adsorção-eluição e precipitação por polietilenoglicol (PEG 6000), e purificação com fluorocarbono (Freon, Dupont Co.) para eliminação prévia dos inibidores da reação de RT-PCR. A extração do ácido nucleico viral, foi realizada com uma mistura de isotiocianato de guanidina e fenol (TRIzol ®, Gibco) e clorofórmio, sendo completada com uma purificação em coluna spin, com membrana de polissulfona (Millipore,Corp.). A reação de RT-PCR foi realizada em uma única etapa, utilizando o kit \'\'Super Script ONE-STEP RT-PCR System\'\'® (Gibco). A reação de amplificação enzimática revelou, por eletroforese em gel de agarose (2%), corada com brometo de etídio, produtos de 123 bp, em 3 (23%) dos 13 lotes de amostras coletadas durante o verão de 1998, sendo verificadas em uma delas por imunomicroscopia eletrônica (IME),partículas de SRSV. Os produtos encontrados pertencem ao grupo genômico G2(Snow Mountain e outros vírus). A aplicação deste método possibilitou pela 1ª vez a detecção molecular de SRSVs no Brasil em ostras naturalmente contaminadas. / The Small Round Structured Viruses (SRSVs) have been associated with gastroenteritis in humans, and were detected in extracts of oysters Crassostrea spp., by Reverse Transcriptase Polymerase Chain Reaction (RT - PCR). Thirteen lots of samples, originated from different regions of the State of São Paulo, were processed for viral particles extraction. The samples, containing 10 g of stomach and diverticula, were extracted by adsorption - elution and polyethylene glycol (PEG 6000) precipitation, and fluorocarbon (Freon, Dupont Co.) was used for purification and previous elimination of RT - PCR inhibitors. For the viral nucleic acid extraction, guanidine isothiocyanate - phenol mixture (TrizolÒ, Gibco ) and chloroform were used, followed by a polysulfone spin column (Millipore, Corp.) purification. The RT-PCR was done in one step, using the \'\'Super Script ONE-STEP RT-PCR System\'\'® (Gibco). The enzimatic amplification reaction run in 2% agarose gel electrophoresis stained with ethidium bromide, revealed 123 bp products in 3 (23%) of the 13 lots of samples collected during the summer of 1998. One of these three samples was positive for SRSV particles by Immune Electron Microscopy (IEM). These 3 products were classified in the Genogroup G2 (Snow Mountain and others viruses). The application of this methodology made it possible to detected, in a molecular basis, the first cases of SRSVs in environmentally contaminated oysters in Brazil.

Vírus causadores de gastroenterite, descritos como pequenos vírus de estrutura arredondada (Small Round Structured Viruses-SRSV), foram detectados em extratos de ostras Crassostrea spp., coletadas em regiões distintas do litoral do Estado de São Paulo, utilizando a Reação em Cadeia por Polimerase com transcrição reversa (RT-PCR).Treze lotes de amostras, contendo 10 g de estômagos e divertículos, foram processados para extração e concentração das partículas virais, mediante adsorção-eluição e precipitação por polietilenoglicol (PEG 6000), e purificação com fluorocarbono (Freon, Dupont Co.) para eliminação prévia dos inibidores da reação de RT-PCR. A extração do ácido nucleico viral, foi realizada com uma mistura de isotiocianato de guanidina e fenol (TRIzol ®, Gibco) e clorofórmio, sendo completada com uma purificação em coluna spin, com membrana de polissulfona (Millipore,Corp.). A reação de RT-PCR foi realizada em uma única etapa, utilizando o kit \'\'Super Script ONE-STEP RT-PCR System\'\'® (Gibco). A reação de amplificação enzimática revelou, por eletroforese em gel de agarose (2%), corada com brometo de etídio, produtos de 123 bp, em 3 (23%) dos 13 lotes de amostras coletadas durante o verão de 1998, sendo verificadas em uma delas por imunomicroscopia eletrônica (IME),partículas de SRSV. Os produtos encontrados pertencem ao grupo genômico G2(Snow Mountain e outros vírus). A aplicação deste método possibilitou pela 1ª vez a detecção molecular de SRSVs no Brasil em ostras naturalmente contaminadas. / The Small Round Structured Viruses (SRSVs) have been associated with gastroenteritis in humans, and were detected in extracts of oysters Crassostrea spp., by Reverse Transcriptase Polymerase Chain Reaction (RT - PCR). Thirteen lots of samples, originated from different regions of the State of São Paulo, were processed for viral particles extraction. The samples, containing 10 g of stomach and diverticula, were extracted by adsorption - elution and polyethylene glycol (PEG 6000) precipitation, and fluorocarbon (Freon, Dupont Co.) was used for purification and previous elimination of RT - PCR inhibitors. For the viral nucleic acid extraction, guanidine isothiocyanate - phenol mixture (TrizolÒ, Gibco ) and chloroform were used, followed by a polysulfone spin column (Millipore, Corp.) purification. The RT-PCR was done in one step, using the \'\'Super Script ONE-STEP RT-PCR System\'\'® (Gibco). The enzimatic amplification reaction run in 2% agarose gel electrophoresis stained with ethidium bromide, revealed 123 bp products in 3 (23%) of the 13 lots of samples collected during the summer of 1998. One of these three samples was positive for SRSV particles by Immune Electron Microscopy (IEM). These 3 products were classified in the Genogroup G2 (Snow Mountain and others viruses). The application of this methodology made it possible to detected, in a molecular basis, the first cases of SRSVs in environmentally contaminated oysters in Brazil.

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