Università degli Studi di Padova Istituto Zooprofilattico Sperimentale delle Venezie, Dipartimento di Scienze Biomediche Comparate Dipartimento di Sanità Pubblica, Patologia Comparata ed Igiene Veterinaria SCUOLA DI DOTTORATO DI RICERCA IN SCIENZE VETERINARIE INDIRIZZO SANITA’ PUBBLICA E PATOLOGIA COMPARATA CICLO XXIV INNOVATIVE TECHNOLOGIES FOR THE DETECTION AND CONTROL OF RABIES Direttore della Scuola : Ch.mo Prof. Massimo Morgante Coordinatore d’indirizzo: Ch.mo Prof. Mauro Dacasto Supervisore :Ch.mo Prof. Massimo Castagnaro Dottorando : Paola De Benedictis Innovative technologies for the detection and control of rabies Paola De Benedictis - PhD Thesis RIASSUNTO.................................................................................................................................................................................................... 5 SUMMARY ...................................................................................................................................................................................................... 7 1 LITERATURE REVIEW ......................................................................................................................................................................... 9 1.1 ETIOLOGY ................................................................................................................................................................................... 10 1.1.1 The family Rhabdoviridae ....................................................................................................................................... 10 1.1.2 The genus Lyssavirus ................................................................................................................................................ 10 1.1.3 The rabies virus (RABV) .......................................................................................................................................... 11 1.1.4 The genes and proteins of the rabies virus .................................................................................................... 12 1.1.4.1 The nucleoprotein ............................................................................................................................................ 13 1.1.4.2 The phosphoprotein ....................................................................................................................................... 14 1.1.4.3 The matrix protein ........................................................................................................................................... 14 1.1.4.4 The glycoprotein ............................................................................................................................................... 14 1.1.4.5 The RNA polymerase ...................................................................................................................................... 15 1.1.4.6 G-L intergenic region ...................................................................................................................................... 15 1.2 RABIES PATHOGENESIS ..................................................................................................................................................... 16 1.3 SIGNS AND SYMPTOMS ....................................................................................................................................................... 17 1.3.1 Symptoms in animals ................................................................................................................................................ 17 1.3.2 Symptoms in humans ................................................................................................................................................ 17 1.4 EPIDEMIOLOGY ....................................................................................................................................................................... 19 1.4.1 Rabies phylogeography ............................................................................................................................................ 19 1.4.2 RABV clades .................................................................................................................................................................... 20 1.5 DIAGNOSIS ................................................................................................................................................................................. 23 1.5.1 Recommended classic diagnostic techniques ............................................................................................... 23 1.5.1.1 Fluorescent antibody test ............................................................................................................................ 23 1.5.1.2 Mouse Inoculation Test and Rabies Tissue Culture Infection Test ........................................ 24 1.5.2 Other recommended methods .............................................................................................................................. 25 1.5.2.1 ELISA-based methods .................................................................................................................................... 25 1.5.3 Methods awaiting further standardisation .................................................................................................... 25 1.5.3.1 Direct Rapid Immunohistochemical Test ............................................................................................ 25 1.5.3.2 Immunochromatographic techniques ................................................................................................... 26 1.5.3.3 Molecular-based methods ........................................................................................................................... 26 1.5.3.4 Internal Amplification Controls ................................................................................................................ 27 1.5.3.5 Reverse-transcriptase polymerase chain reaction (RT-PCR ..................................................... 27 1.5.3.6 Real time RT-PCR ............................................................................................................................................. 28 1.5.3.7 Nucleic acid sequence-based amplification (NASBA ..................................................................... 28 1.5.3.8 Loop-Mediated Isothermal Amplification (LAMP ........................................................................... 29 1.5.3.9 Microarray detection of lyssaviruses ..................................................................................................... 29 1.6 FUNDAMENTALS OF APPLIED EXPERIMENTAL TECHNIQUES .................................................................... 30 1.6.1 Sequencing ...................................................................................................................................................................... 30 1.6.1.1 Sanger sequencing ........................................................................................................................................... 30 1.6.1.2 Pyrosequencing ................................................................................................................................................. 30 1.6.2 Phylogenetic techniques .......................................................................................................................................... 30 1.6.2.1 Distance methods ............................................................................................................................................. 31 1.6.2.2 Maximum parsimony ..................................................................................................................................... 31 1.6.2.3 Maximum Likelihood methods .................................................................................................................. 32 1.6.2.4 Estimating the reliability of a phylogenetic tree .............................................................................. 32 1 Innovative technologies for the detection and control of rabies Paola De Benedictis - PhD Thesis 1.6.2.5 Bayesian analysis.............................................................................................................................................. 32 1.6.3 Evolutionary models .................................................................................................................................................. 33 1.7 OBJECTIVES OF THE STUDY ............................................................................................................................................. 34 2 APPLICATION OF MOLECULAR METHODS TO IMPROVE RABIES DIAGNOSIS ............................................ 35 2.1 DEVELOPMENT OF A ONE STEP RT-PCR FOR RABIES DIAGNOSIS IN ANIMALS AND HUMANS 36 2.1.1 Background..................................................................................................................................................................... 36 2.1.2 Materials and methods ............................................................................................................................................. 36 2.1.2.1 Viruses and bacterial strains ...................................................................................................................... 37 2.1.2.2 RNA extraction