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Provides a comprehensive guide to laboratory techniques for research and diagnosis in rabies, and the production of vaccine and immunoglobulin. Now in its fourth edition, this standard reference work has been substantially revised and expanded following more than two decades of widespread use of the previous edition. The book has six parts. The first provides a general overview of current techniques, including safety precautions, and summarizes the characteristics and molecular biology of the rabies virus. The second and third parts evaluate diagnostic and research techniques, including those involving the use of enzyme-linked immunosorbent assays (ELISAs), cell cultures, monoclonal antibodies and the polymerase chain reaction.

The production of rabies vaccines for human and veterinary use is considered in the fourth part, which includes 23 new chapters dealing with vaccines prepared in cell cultures (including continuous cell lines) and recombinant vaccines. Techniques for evaluating the safety and potency of rabies vaccines are then described, including in vitro tests for in-process control. The final part covers the production of human and equine antirabies serum and immunoglobulin. Further practical advice is given in the appendices, which describe techniques for the collection and shipment of brain specimens for rabies diagnosis, preparation of rabies conjugates, and calculation of virus titres and antirabies serum, immunoglobulin and vaccine potency.

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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. Meslin ; M. M. Kaplan = 9
    Introduction = 9
    Diagnostic procedures for antigen detection = 10
    Tests for the determination of rabies antibody = 13
    Potency tests = 14
    Reserch techniques = 15
    Conclusion = 16
    References = 16
  Chapter 3 Characteristics and molecular biology of the rabies virus / N. Tordo = 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 / E. S. Tierkel ; P. Atanasiu = 55
    Dissection of the brain = 55
    Preparation of slides = 55
    The Negri body : differential diagnosis = 59
    The mouse inoculation test = 60
    Annex Preparation of sellers' stain = 62
  Chapter 5 Histopathological diagnosis / P. L e ´ pine ; P. Atanasiu = 66
    Removal of the brain nd preparation of tissue samples for examination = 68
    Embedding, staining and examination for Negri bodies = 77
  Chapter 6 The mouse inoculation test / H. Koprowski = 80
    Choice of mice = 80
    Preparation of suspect material for inoculation = 81
    Inoculation of mice = 83
    Observation of inoculated mice = 84
    Further passages of infected material = 85
    Removal of the brain = 85
    Complications = 86
  Chapter 7 The fluorescent antibody test / D. J. Dean ; M. K. Abelseth ; P. Atanasiu = 88
    Principle = 88
    Materials and methods = 89
    Discussion = 93
  Chapter 8 Virus isolation in neuroblastoma cell culture / W. A. Webster ; G. A. Casey = 96
    Rabies tissue ­ culture infection test (RTCIT)
    References = 101
    Annex 1 Media = 102
    Annex 2 Avidin ­ biotin staining method = 103
  Chapter 9 Rapid rabies enzyme immunodiagnosis (RRID) for rabies antigen detection / H. Bourth ; P. Perrin = 105
    Introduction = 105
    Method = 105
    Evaluation of the technique = 111
    References = 111
    Annex Preparation of regents = 112
  Chapter 10 Cell culture of rabies virus / A. A. King = 114
    Susceptible cells, cell lines and strains = 114
    Methods of virus propagation = 115
    Cytopathology = 117
    Persistent infection = 117
    Virus in infected cells = 118
    Appliccation of cell ­ culture methods = 119
    References = 122
Part Ⅲ. Special diagnostic and research techniques = 131
  Chapter 11 Techniques for the production, screening and characterization of monoclonal antibodies / M. Lafon = 133
    Immunization of animals = 133
    Myelomas = 134
    Fetal calf sera = 134
    Fusion = 134
    Screening of hybridoma supernatants = 136
    Cloning of hybridomas by limiting dilution = 139
    Production of large amounts of monoclonal antibodies = 139
    Freezing and thawing of hybridomas = 140
    Characterization of monoclonal antibodies = 141
    Use of monoclonal antibodies = 142
    References = 142
    Annex Dulbecco's modified Eagle's medium (DMEM) = 144
  Chapter 12 Monoclonal antibodies for the identification of rabies and non ­ rabies lyssaviruses / J. S. Smith ; A. A. King = 145
    Materials and methods = 145
    Immunofluorescence tests using MAb ­ RNPs = 147
    Antigenic analysis using MAb ­ Gs = 147
    Applications = 148
    Discussion = 155
    References = 155
  Chapter 13 The polymerase chain reaction (PCR) technique for diagnosis typing and epidemiological studies of rabies / N. Tordo ; D. sacramento ; H. Bourhy = 157
    Introduction = 157
    Amplification of the rabies transcripts = 157
    diagnosis = 159
    Typing and molecular epidemiological studies = 164
    References = 169
    Annex Preparation of buffers and reagents = 170
  Chapter 4 Techniques for the purification of rabies virus, its subunits and recombinant products / B. Dietzschold = 175
    Introduction = 75
    Purification of rabies virus particles = 175
    Purification of rabies virus subunits and structural proteins under non ­ denaturing conditions = 176
    Purification of rabies virus proteins under denaturing conditions = 178
    References = 179
    Annex Preparation of reagents = 179
  Chapter 15 A rapid fluorescent focus inhibition test (RFFIT) for determiningrabies virus ­ neutralizing antibody / J. S. Smith ; P. A. Yager ; G. M. Baer = 181
    Standard prodedure = 181
    Calculation of virus ­ neutralizing antibody titres = 185
    Alternative test procedures = 186
    Interpretation of results = 188
    Annex 1 Growth media for MNA and BHK­21 S13 cells = 190
    Annex 2 Calculation of titres = 191
  Chapter 16 An in vitro virus neutralization test for rabies antibody / C. V. Trimarchi ; R. D. Rudd ; M. Safford, Jr = 193
    Method = 193
    Interpretation of results = 198
    References = 198
  Chapter 17 Competitive ELISA for the detection of rabies virus ­ neutralizing antibodies / L. D. Elmgren ; A. I. Wanderler = 200
    Method = 201
    Interpretation of results = 204
    References = 204
    Annex 1 Preparation of buffers and reagents = 206
    Annex 2 Conjugation of monoclonal antibodies = 207
    Annex 3 ALISA software = 207
  Chapter 18 Electron microscopy / K. Hummeler ; P. Atanasiu = 209
    Structural studies = 209
    studies of morphogenesis = 209
    Annex Medium for agarose : Eagle's basal medium (EBM) = 216
Part Ⅳ. Methods of vaccine production = 219
  Section A. Brain ­ tissue vaccines = 221
    Chapter 19 General considerations in theproductionand use of braintissue and purified chicken ­ embryo rabies vaccines for human use / F. ­X. Meslin ; M. M. Kaplan = 223
      Introduction = 223
      Adverse effects of brain ­ tissue vaccines = 224
      Recent developments in brain ­ tissue vaccine production = 226
      References = 228
    Chapter 20 β­Propiolactone ­ inactivated sheep brain vaccine / H. Singh = 234
      Composition = 234
      Preparation of the seed virus = 234
      Preparation of the vaccine = 235
      Quality control tests = 237
      Biochemical test = 238
      Preparation of standard vaccine = 240
      Dosage schedule = 240
      References = 241
      Annex 1 Preparation of0.5 mol／1 sodium ­ potassium phosphate buffer, pH 7.6 = 241
      Annex 2 Preparation of 0.005 mol／1 phosphate ­ buffered saline (PBS), Ph7.0 = 242
      Annex 3 Preparation of stabillzer for rabies vaccine, pH7.2 = 242
    Chapter 21 Suckling ­ mouse brain vccine / A. M. Diaz = 243
      Formula = 243
      Preparation of the inoculum = 243
      Inoculation and harvest = 244
      Preparation of the vaccine = 245
      Control tests = 245
      Expiry date = 247
      References = 247
      Annex 1 Ultraviolet irradiation for inactivation of vaccines = 248
      Annex 2 Preparation of stabilizer solutions = 250
  Section B. Embryonating egg vaccines = 251
    Chapter 22 Purified duck ­ embryo vaccine for humans / R. Gl ￦ddotu ck = 253
      Preparation of the vaccine = 253
      Control tests = 257
      Expiry date = 258
      References = 258
      Annex Preparation of stabilizing medium = 258
    Chapter 23
    Chicken ­ embryo vaccine for dogs / H. Koprowski = 260
      Preparation of the vaccine = 260
      Control tests = 265
      References = 266
      Annex Preparation of stabilizing solution, pH 7.6 = 266
  Section C. Cell ­ culture vaccines = 269
    Chapter 24 Cell ­ culture vaccines for human use : general considerations / K. G. Nicholson = 271
      Human diploid cell vaccine = 271
      Other cell ­ culture vaccines = 271
      Safety = 272
      Efficacy = 274
      Economical post ­ exposure treatment regimens = 275
      Pre ­ exposure immunization = 276
      References = 276
    Chapter 25 Vaccine for humans prepared in human diploid cells / R. Branche = 280
      Preparation of the vaccine = 280
      Control tests = 281
      Conclusion = 283
      References = 283
      Annex Flow chart for the production of HDC vaccine using the MRC5 cell strain = 284
    Chapter 26 Purified Vero cell vaccine for humans / B. Montagnon ; B. Fanget = 285
      Cell cultures = 285
      Preparation of the vaccine = 286
      Control tests = 287
      Expiry date = 288
      References = 288
    Chapter 27 Purified chick ­ embryo cell vaccine for humans / R. Barth ; V. Franke = 290
      History = 290
      Preparation of the vaccine = 290
      Control tests = 292
      Administration of the vaccine = 293
      Expiry date = 293
      Laboratory tests = 294
      References = 294
    Chapter 28 Fetalrhesus monkey lung diploid cell vaccine for humans / R. Barth ; V. Franke = 297
      History = 297
      Preparation of the vaccine = 297
      Control tests = 298
      Expiry date = 299
      Administration of the vaccine = 299
      Laboratory tests = 299
      References = 299
    Chapter 29 Dog kidney cell vaccine for humans / R. Barth ; V. Frnke ; G. Van Steenis = 301
      Preparation of the vaccine = 301
      Control tests = 302
      Administration of the vaccine = 303
      References = 303
      Annex Preparation of medium 199 = 303
    Chapter 30 Primary hamster kidney cell vaccine for humans / R. Barth ; V. Franke ; F. T. Lin = 306
      Preparation of the vaccine = 306
      Control tests = 307
      Expirydate = 308
      Administration of the vaccine = 308
      References = 308
      Annex Hanks' balanced salt solution = 309
    Chapter 31 Vnukovo­32 primary hamster kidney cell vaccines for humans / R. Barth ; V. Franke ; M. A. Selimov = 310
      Preparation of the vaccines = 310
      Control test = 312
      Administration of the vaccines = 312
      References = 312
    Chapter 32 Cell ­ culture vaccines for veterinary use / P. Reculard = 314
      Substrates for the production of seed virus and vaccine = 314
      Preparation of the vaccines = 316
      Manufacturing requirements for cell ­ culture rabies vaccines and recommendations for their use = 318
      Planning a facility for the production of rabies vaccine for veterinary use = 321
      References = 331
  Section D. Genetically engineered vaccines = 339
    Chapter 34 General considerations in the use of recombinant rabies vaccines for oral immunization of wildlife / C. E. Rupprecht ; C. A. Hanlon ; H. Koprowski = 341
      References = 344
    Chapter 35 Exprssion of rabies proteins using prokaryotic and eukaryotic expression systems / B. Dietzshold = 347
      Prokaryotic expression systems = 347
      Eukaryotic expression systems = 347
      References = 350
Part Ⅴ. Vaccine safety and tests for potency and antigen quantification = 353
  Chapter 36 General considerations in testing the safety and potency of rabies vaccines / P. Sizaret = 355
    References = 358
  Chapter 37
  The NIH test for potency / L. A. Wilbur ; M. F. A. Aubert = 360
    Standard test = 360
    Modified NIH test = 365
    References = 368
    Annex Preparation of Challenge Virus Standard (CVS) diluent, pH 7.6 = 368
  Chapter 38 Habel test for potency / K. Habel = 369
    Standard test = 369
    Modified Habel test = 370
    Annex Calculating 50% end ­ point dilutions by the method of Reed & Muench = 371
    Chapter 39 Guinea ­ pig test for chicken ­ embryo vaccine / H. Koprowski = 374
    Immunization procedure = 374
    Preparation of challenge material = 374
    Challenge of the guinea ­ pigs = 376
    Interpretation of results = 376
  Chapter 40 Single radial immunodiffusion test for the determination of the glycoprotein content of inactivated rabies vaccines / M. Ferguson = 378
    Principle = 378
    Materials = 378
    Method = 379
    Interpretation of results = 381
    References = 382
    Annex Preparation of Dulbecco's phosphate ­ buffered saline solution A (PBSA), pH 7.2 = 382
  Chapter 41 Enzyme ­ linked immunosorbent assay (ELISA) for the determination of the glycoprotein content of rabies vaccines / P. Perrin ; M. Lafon ; P. Sureau = 383
    Principle = 383
    Preparation of antibodies = 383
    Precautions = 383
    Sensitization of microtitration plates = 384
    Assay procedure = 384
    Interpretation of results = 386
    Evaluation of in vitro potency = 387
    References = 387
  Chapter 42 The Essen ­ELISA for the determination of the glycoprotein content of inactivated cell ­ culture rabies vaccines / O. Thraenhart = 389
    Principle = 389
    Method = 389
    Evaluation of results = 391
    References = 393
    Annex Preparation of buffers and reagents = 393
  Chapter 43 The modified antibody ­ binding test for in vitro quantifiction of rabies virus antigen in inactivated rabies vaccines / R. Barth = 394
    Principle = 394
    Method = 394
    Interpretation of results = 395
    Evaluation of the test = 395
    References = 396
Part Ⅵ. Antirabies serum and immunoglobulin = 399
  Chapter 44 Production of antirabies serum of equine origin / T. Luekrajang ; J. Wangsai ; P. Phanuphak = 401
    Introduction = 401
    Method = 401
    Factors affecting the production of ERIG = 402
    Safety = 403
    References = 403
  Chapter 45 Purificatin techniques for heterologous rabies antiserum / R. Gl ￦ddotu ck ; D. Labert = 405
    Preservation and storage of serum or plasma = 405
    Purification by enzyme treatment and heat denaturation = 406
    Purification by precipitation using ethacridine lactate and ethanol = 408
    Stabilization and preservation of purified HRIG = 409
    Standardization of the final product = 409
    References = 409
    Annex Preparation of buffers = 410
  Chapter Production of human rabies immunoglobulin / P. Fournier ; R. K. Sikes = 411
    Introduction = 411
    Formula = 411
    Source and shipment of blood = 411
    Reagents = 412
    Technique = 412
    Disadvantages = 415
    References = 415
    Annex Preparation of reagents = 416
  Chapter 47 Potency test for antirabies serum and immunoglobulin / E. A. Fitzgerald = 417
    Principle = 417
    preparation and titration of challenge virus = 417
    Serum virus neutralization = 418
    Interpretation of results = 419
    Currently used potency tests = 420
    References = 421
Appendices = 423
Appendix 1 Simple technique for the collection and shipment of brain specimens for rabies diagnosis / J. Brrat = 425
Appendix 2 Techniques for the preparation of rabies conjugates / P.Perrin = 433
Appendix 3 Methods for the calculation of titre / M. F. A. Aubet = 445
Appendix 4 Addresses of international institutions for technical cooperation in rabies control = 460
Index = 469