WHO/IVB/08.17 ORIGINAL: ENGLISH Manual of rotavirus detection and characterization methods Immunization, Vaccines and Biologicals WHO/IVB/08.17 ORIGINAL: ENGLISH Manual of rotavirus detection and characterization methods Immunization, Vaccines and Biologicals The Department of Immunization, Vaccines and Biologicals thanks the donors whose unspecified financial support has made the production of this document possible. This document was produced by the Expanded Programme on Immunization of the Department of Immunization, Vaccines and Biologicals Ordering code: WHO/IVB/08.17 Printed: October 2009 This publication is available on the Internet at: www.who.int/vaccines-documents/ Copies of this document as well as additional materials on immunization, vaccines and biologicals may be requested from: World Health Organization Department of Immunization, Vaccines and Biologicals CH-1211 Geneva 27, Switzerland • Fax: + 41 22 791 4227 • Email: [email protected] • © World Health Organization 2009 All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791 3264; fax: +41 22 791 4857; email: [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; email: [email protected]). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication. Printed by the WHO Document Production Services, Geneva, Switzerland ii Contents Acknowledgements .............................................................................................................v Using this manual ............................................................................................................ vii 1. Introduction and overview ......................................................................................1 1.1 Background ..........................................................................................................1 1.2 Rotavirus Detection ............................................................................................2 1.3 Rotavirus Characterization .................................................................................4 1.4 Safety precautions .............................................................................................16 1.5 Specimen requirements .....................................................................................17 1.6 Shipping ..............................................................................................................17 2. Laboratory Procedures:Rotavirus Detection .....................................................18 2.1 Virus cultivation .................................................................................................19 2.2 Detection of rotavirus in cell culture by IF .....................................................21 2.3 Electron microscopy (EM) ...............................................................................22 2.4 PAGE and silver staining .................................................................................23 3. Rotavirus characterization: Serological Methods .............................................31 3.1. Serotyping and subgrouping with monoclonal antibodies ............................31 4. Rotavirus characterisation: Molecular methods ................................................47 4.1. Method 1: Manual RNA extraction from stool .............................................47 4.2 Method 2: RNA extraction using hydroxyapatite .........................................49 4.3 Method 3: RNA Extraction Using RNAID ..................................................51 4.4 Method 4: RNA extraction using Trizol ........................................................53 4.5 Method 5: RNA extraction using the CTAB method ...................................54 4.6 Method 6: RNA extraction using the Boom method .....................................55 4.7 Method 7: RNA extraction from cell lysates ..................................................56 4.8 Method 8: G Genotyping ..................................................................................59 4.9 Method 9: P Genotyping .................................................................................63 4.10 Method 10: G Genotyping ................................................................................66 4.11 Method 11: P Genotyping ...............................................................................70 4.12 Method 12: G Genotying .................................................................................73 4.13 Method 13: P Genotyping ...............................................................................77 4.14 Method 14: G genotyping ................................................................................80 4.15 Method 15: P Genotyping ...............................................................................85 4.16 Method 16: G and P genotyping ......................................................................91 4.17 Trouble shooting ...............................................................................................99 5. References ...............................................................................................................100 iii Appendix 1: Oligonucleotide primers ........................................................................104 Appendix 2:Cloning and sequencing methods .........................................................108 Appendix 3: Preparation of reagents .........................................................................132 Appendix 4:Suppliers ....................................................................................................138 Appendix 5:Worksheets ................................................................................................143 iv Acknowledgements This document was produced for the Expanded Programme on Immunization of the Department of Immunization, Vaccines and Biologicals, WHO by the Laboratory Directors of the following Regional Rotavirus Laboratories and the WHO Rotavirus Collaborating Centers: Jon Gentsch WHO Collaborating Center (US) Gastroenteritis and Respiratory Viruses Laboratory Branch Division of Viral Diseases National Center for Immunization and Respiratory Diseases Coordinating Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, US Jim Gray and Miren Iturriza-Gómara European Regional Rotavirus Laboratory Enteric Virus Unit Virus Reference Department Centre for Infections Health Protection Agency London, UK John Klena Eastern Mediterranean Regional Rotavirus Laboratory Enteric Diseases Research Program U.S. Naval Medical Research Unit No. 3 Cairo, Egypt Carl Kirkwood WHO Collaborating Center (Australia) Enteric Virus group Murdoch Childrens Research Institute, Royal Children’s Hospital Victoria, Australia George Armah West African Rotavirus Regional Laboratory Department of Electron Microscopy and Histopathology Noguchi Memorial Institute for Medical Research University of Ghana, Legon, Ghana v Nicola A. Page MRC Diarrhoeal Pathogens Research Unit (DPRU) University of Limpopo (Medunsa Campus) PO Box 173, Medunsa 0204, South Africa The following members of the “Rotavirus Experts External Advisory Group” kindly reviewed and provided helpful comments on the document: Kari Johansen - European Centre for Disease Prevention and Control, Ana Maria Bispo - PAHO, Duncan Steele - PATH and Timo Vesikari - University of Tampere Medical School. vi Using this manual This manual has been prepared by staff of the WHO Rotavirus Collaborating Centers and Regional Laboratories throughout the world and is a comprehensive collection of methods for the detection and characterisation of rotaviruses. The manual should be used in conjunction with the training provided in a Collaborating Centre or Regional Laboratory and contact with that centre should be maintained in order to provide quality assurance and frequent updates of methods. For some of the techniques described, a single method is provided, indicating general consensus among the network of rotavirus laboratories. Where multiple methods