Laboratory techniques in rabies FOURTH EDITION Edited by F.-X. Meslin M. M. Kaplan H. Koprowski Chief Former Director Director Veterinary Public Health Research Promotion Center for Neurovirology Division of Communicable and Development Jefferson Cancer Institute Diseases World Health Organization Thomas Jefferson University World Health Organization Geneva, Switzerland Philadelphia Geneva, Switzerland PA, USA World Health Organization Geneva 1996 WHO Library Cataloguing ip Pbbicato~iData Laboratory tec?r?ques in rabies edited by F -X Mes in V h; Kapdri H Koprnwsk - 4th ed 1 Rabies diagnosis laboratorv marLals 2 Haoies vaccine I Mesin F.-X l1 Kaplan. M.M. lli.tioprowsk!, H ISBN 92 4 154,479 1 (NLM Classification WC 550) The Word Health O~gaiizat~onwelcomes requests for permission to reproduce or translate its poblica- tions in part or in full Applicaliiins ar?ri enquiries should be addressed !o the Office of Publ~cationsWorld Health Oryariiiation Geneva Switzerland whicti will be glad to provide the latest ir~forrnationon any changes made to the text palls for new editions, and reprints arid lranslatlons already available @World Health Organization 1996 Publicat~ons of the World Healtil Organiratioil enjoy copyright protection in accordaricc with the provisions of Protocol 2 of the Universal Copyrigtit Conbention All rights reserved The oesgnations ernp:oyed ard the prese.itaton of tt~?material in this publication do not irnply the express10:i ui ariy op'lior: wiiatsoeve~on the part of thr Sccretaria: of the World lieallh Organization concer~ing!?e legal s:a!us o: any country terrilory citv or area or of its authorities, or concerning the delimitation oi is frontiers or DO~rlddrleS The -:lention of specitic companies or of certain rnan~fac:~irers'products ooes no: imply that they are endorsed or recommended by :he Worla Heaitb Orgar~7ation~r, preference to otners of a sirnilar natdre that are no! mentioled Errors and owissior3s exceo:ed, tqe names of propfietary pronucts are distinguisti~~iby iiiitial capi!al leiters The authors aloqe are responsible foi the vleivs expressed in this uublicatiori Where the designation ',courtry of area ' appears in the headings of tables. I? covers countrles, te:rltories cities or areas Typeset il India Printed in Finiarid 93~9814-Macm1llars!Vam~ala8500 Contents Preface xiii List of acronyms and abbreviations used in this book xv Part I. General considerations 1 Chapter 1 Safety precautions in handling rabies virus (M M Kaplan) 3 Properties of the virus 3 Pathogenesis 3 Laboratory precautions 4 Treatment of wounds 5 Pre-exposure immunization 6 References 7 Chapter 2 An overview of laboratory techniques in the diagnosis and prevention of rabies and in rabies research (F.-X Mes/in & MM��� 9 Introduction 9 Diagnostic procedures for antigen detection 10 Tests for the determination of rabies antibody 13 Potency tests 14 Research techniques 15 Conclusion 16 References 16 Chapter 3 Characteristics and molecular biology of the rabies virus ( N. Torda) 28 Introduction 28 Morphology and structure 28 Functional analysis of the infection 32 Molecular biology of the rabies virus 36 Evolution of the rabies virus 43 References 45 Part II. Routine laboratory procedures 53 Chapter 4 Rapid microscopic examination for Negri bodies and preparation of specimens for biological tests 55 (E. S. Tierkel & P. Atanasiu) Dissection of the brain 55 iii LABORATORY TECHNIQUES IN RABIES Preparation of slides Tlie Negri body: differential diagnosis The mouse inoculation test Annex Pre~arationof Sellers' stain Chapter 5 Histopathological diagnosis (P Lepine & P. Atanasiu) 66 Removal of the bra~nand preparation of tissue samples for examination 68 Ernbedciirig, shining and examination for Negri bodies 77 Chapter 6 The lnouse inoculation test (H. Koprowsk~) Choice of mice Preparation of suspect material for rnoculation Inoculation of mice Observation of inoculated mice Further passages of infected material Removal of tile brain Complications Ctiapter 7 The fluorescent antibody test (D J Dean M K Abelseth & P Atanas~u) 88 Principle 88 Materials and methods 89 Discussion 93 Chapter 8 Virus isolation in neuroblastorna cell culture A. Webster & G. A. Casey) 96 Rabies tissue-culture infection test (RTCIT) 97 References 101 Annex l Media 102 Annex 2 Avidin-biotin staining method 103 Chapter 9 Rapid rabies enzyme irnmunodiagnosis (RREID) for rab~es ant~gendetection (H Eourhy & P Perrin) 105 Introduction 105 Method 105 Evaluation of the technique 11 1 References 11 1 Annex Preparation of reagents 112 Chapter 10 Cell culture of rabies virus (A A King) Susceptible cells, cell lhnes and strains Methods of virus propagation Cytopathology Persistent infection Virus in intected cells Application of cell-culture methods References CONTENTS Part Ill. Special diagnostic and research techniques Chapter 11 Techniques for the production, screering and characteriza- tion of rnonoclonal antibodies (M, Lafon) Immunization of animals Myelomas Fetal calf sera Fusion Screening of hybrdoma supernatants Cloning of hybridomas by lim~lingdili~tion Prodi~ctionof large n~nountsof rnonnclonal antibodies Freezing and thawing of hybridomas Characterization of rnonoclonal antibodies Use of rnonoclonal antibodies References Aniiex Dulbeccos modified Eagle's medium (DMEM) Chapter 12 Monoclonal antibodies for the identification of rabies and non-rabies lyssaviruses (J. S. SmitA & A. A. King) Materials and methods lmmunofliiorescence tests using MAb-RNPs Antigenic analysis using MAb-Gs Applications Discussion References Chapter 13 The polynlerase chain reaction (PCR) technique for diagnosis. typing and epidemiological studies of rabies (N. Tordo, D. Sacrament0 & H. Bourhy) Introduction Amplification of the rabies transcripts Diagnosis Typing and molecular epidemiological studies References Annex Preparation of buffers and reagents Chapter 14 Techniques for the purification of rabies virus, its subunits and recornbinaril products (B. Dietzschold) Introduction Pcrrificatiori of rabies virus particles Purification of rabies virus siibunits and structural proteins under non-denaturing conditions Purification of rabies virus proteins under denaturing coriditions References Annex Preparation of reagents Chapter 15 A rapid fluorescent focus inhibition test (RFFIT) for deter- mining rabies virus-~ieiitralizingantibody (J. S. Smith. P. A. Yager & G. M. Baer) LABORATORY TECHNIQUES IN RABIES Standard procedure Calcu!at~onof virus-neutralizing antibody titres Alternative test procedures Interpretation of results References Annex 1 Growth media for MNA and BHK-21 S13 cells Annex 2 Calculation of titres Chapter 16 An in vltro virus neutralization test for rabies antibody (C. V Triinarchi. R. D. Rudd & M. Saffoid.Jr) Method Interpretation of results References Chapter 17 Competitve ELISA for the detection of rabies virus-neutralizing antibodes (L. D. Eimgren & A. l. Wandeler) Method Interpretation of results References Annex 1 Preparaion of buffers and reagents Annex 2 Conjugaton of monoclona! antibodies Annex 3 ELISA software Chapter 18 Electron microscopy (K. Hummeler & P. Atanasiu) Structural studes Studies of morphogenesis Annex Medium for agarose: Eagle's basal medium (EBM) Part IV. Methods of vaccine production Section A. Brain-tissue vaccines 221 Chapter 19 General consideratons in the production and use of brain- tissue and purified chicken-embryo rabes vaccines for human use (F.-X. Meslin & M. M. Kaplan) 223 Introduction 223 Adverse efTects of brain-tissue vacclnes 224 Recent developments in brain-tssue vaccine production 226 References 228 Chapter 20 P-Propolactone-inactivated sheep brain vaccine (H. Sir7gii) 234 Cornpositon 234 Preparation of the seed vrus 234 Preparation of the vaccine 235 Quality control tests 237 Biochemical tests 238 Preparation of standard vaccine 240 Dosage schedule 240 References 241 Annex 1 Preparation of 0.5 mol/l sodium-potassium phosphate buffer, pH 7.6 24 1 CONTENTS Annex 2 Preparation of 0.05 rnol/l phosphate-buffered saline (PBS), pH 7 0 Annex 3 Preparation of stabilizer for rabies vaccine, pH 7.2 Chapter 21 Suckl~ng-mousebrain vaccine (A M Dlaz) Formilla Preparat~onof the inoculum Inoculation and harvest Preparaton of the vaccine Control tests Expiry date References Annex 1 Ultraviolet irradiation for inactivation of vaccines Annex 2 Pre~arationof stabilizer solutions Section B. Ernbryonating egg vaccines Chapter 22 Purified duck-embryo vaccine for Run~ans(R. Gluck) Preparation of the vaccine Control tests Expiry date References Annex Preparation of stabilizing medium Chapter 23 Chicken-embryo vaccine for dogs (H. Koprowski) Preparation of the v-=ccine Control tests References Annex Preparation of stabilizing solution, pH 7.6 Section C. Cell-culture vaccines Chapter 24 Cell-culture vaccines for Iiunian use general considerations (K G Nicholson) Human diploid cell vaccine Other cell-culture vaccines Safety Efficacy Economical post-exposure treatment regimens Pre-exposure ~mrnun~zation References Chapter 25 Vaccine for humans prepared in human diploid cells (R. Brarictie) Preparation of the vaccine Contl-ol tests Conclusion References Annex Flow chart for the production of HDC vaccine using the MRC5 cell strain LABORATORY TECHNIQUES IN RABIES Chapter 26 Purified Vero cell vaccine for humans (B. Montagnon & B. Fanget) Cell cultures Preparation of the vaccine Control tests Expiry date References Chapter 27 Purified chick-ernbryo cell vaccine for humans [R. Bartti & K Franke) History Preparatiori of the vaccine Control tests Administratioii