Rabies is a fatal but preventable neurotropic disease of potentially all mammals. The disease is caused by lyssaviruses. Rabies is recognized as the 10th most common lethal infectious disease in the world, rendering it one of the most feared zoonotic diseases known to man. Nevertheless, rabies can be prevented by application of pre- or post exposure treatments. Rabies vaccines have been available since the time of Pasteur, more that one hundred years ago. Since, vaccine research focused on the development of safer and more effective vaccines. Topics of current interest in the field of rabies vaccinology were addressed in this study. A primary concern regarding the disease is human mortalities, in the range of 60 000, reported every year. Most of these are linked to exposure to rabid dogs. In addition, a great number of post exposure treatments are administered each year at great costs. Despite availability of efficacious biologics, several factors influence the optimal use and accessibility of these agents in the countries of interest, with cost and availability being the major contributing factors. A proven approach is mass oral vaccination of target animals, such as dogs, which indirectly infers protection to susceptible hosts, including man. Currently available vaccines present several disadvantages of use though, including issues of safety or doubtful stability. Safer but effective alternative vaccines that could be used in oral baits would be valuable. Here the use of two candidate host restricted poxvirus vaccine vectors were explored, particularly also in regard to oral innocuity. The construction, convenient isolation and use of a recombinant Lumpy skin disease virus (Neethling strain) expressing rabies virus glycoprotein in a mouse model were investigated. In addition, a recombinant Modified Vaccinia virus Ankara expressing rabies virus glycoprotein was prepared and tested as a vaccine in mice, dogs and raccoons. In both cases it was clear that the severe attenuation of these viruses did affect the efficacy of the recombinant vaccines in the non-permissive hosts. With the recombinant MVA a clear dosage effect could be shown, and equivalent humoral responses could only be attained at much higher titers of vaccine virus as with replication competent counterparts. Secondly, the cross-protection of rabies vaccines across the spectrum of lyssaviruses was addressed. Lyssaviruses can be divided into two groups based on sequence analysis and pathogenesis. Viruses belonging to the so-called phylogroup II, are the Mokola, Lagos and West Caucasian Bat viruses. Classic rabies biologics fail to fully protect against the viruses attributed to a lack of cross-neutralization. Here, cross-protection and cross-reactive immune responses induced by recombinant vaccinia viruses expressing rabies, Mokola or West Caucasian Bat virus glycoproteins, in single or dual combinations, were investigated. As expected, there was a lack of cross-protection of rabies and Mokola glycoprotein vaccines. There was also a clear lack of cross-protection of West Caucasian Bat virus glycoprotein vaccine and rabies and Mokola viruses. The dual antigen expressing vaccines did not appear to offer any additional protective effect in the tested model. The Mokola virus glycoprotein vaccines induced neutralizing antibody responses that significantly cross-neutralized Lagos Bat virus. / Thesis (PhD (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

No description available.

No description available.

A two-part study was undertaken to investigate and model the characteristics of rabies epizootics in wildlife populations. Initial analysis of temporal and spatial relationships between reported rabies cases in four ecologically divergent counties was conducted. No single pattern of report incidence was found to be characteristic of all counties. Concentration of rabies reports near foci of human population was the only common trend, confirming previous studies of the dependence of rabies reporting upon human population density. Topography partly determines the pattern of hum:an habitation, thereby influencing rabies report data. A computer model of wildlife rabies epizootics was written for an IBM 360/65-360/50. Based on a generalized simulation language, GASP II, this model may be used to simulate the effects of proposed rabies control.strategies on present and future epizootics. Demonstrations of techniques for simulating control by popµlation reduction, vaccination, and sterilization were presented. Statements of model limitations and suggestions for future improvements were included in the discussion of project results. / Master of Science

O presente trabalho visou estudar o comportamento biológico em camundongos de 10 amostras de vírus da raiva isoladas de morcegos hematófagos e não-hematófagos, no Estado do Rio de Janeiro, sua caracterização genética quanto aos genes N e G, além da resposta de camundongos vacinados com a vacina anti-rábica produzida pela replicação da amostra Pitman-Moore em cultivo celular, frente ao desafio com estas amostras virais. As passagens sucessivas em camundongos determinaram uma redução do período de incubação e a obtenção de amostras virais com altos títulos. A vacina anti-rábica utilizada na imunização dos camundongos ofereceu proteção em mais de 80% dos camundongos vacinados com a diluição 1:5 da vacina, frente à maioria das amostras. A análise filogenética do gene da proteína N apresentou um padrão de agregação dividido em variante de morcego hematófago e variante de morcego insetívoro, com diferenças filogenéticas entre as variantes de morcego hematófago isoladas na Região Noroeste do Estado do Rio de Janeiro e aquelas isoladas nas Regiões Metropolitana e Sul do Estado. A substituição do resíduo ácido aspártico por ácido glutâmico na posição 118, encontradas na caracterização genética da proteína G das amostras 704/97 e 151/98, permite inferir que esta posição esteja relacionada à antigenicidade viral. Não foram observadas diferenças genéticas temporais entre as amostras estudadas. / In the present study we analyzed the biological behavior in mice of 10 rabies virus samples isolated from haematophagous and non-haematophagous bats in Rio de Janeiro State, its genetic characterization from genes N and G, and we also studied the response of mice vaccinated with cell culture rabies vaccine, produced with the Pitman-Moore strain, after viral challenge with bat rabies virus samples. Viral passages in mice resulted in reduced incubation period and high virus titrers. The vaccine used conferred protection in more than 80% of the mice vaccinated with 1:15 vaccine dilution, after viral challenge. N gene genetic analysis divided the samples in haematophagous bat strains and insectivorous bat strains, with phylogenetic differences between samples isolated in Northeast Region and those isolated in the Metropolitan and South Regions of Rio de Janeiro State. The substitution of an aspartic acid to a glutamic acid found in the position 118 of G gene genetic characterization from samples 704/97 and 151/98 seems to be related to viral antigenicity. There were no time-related genetic differences between the studied samples.

O presente trabalho visou estudar o comportamento biológico em camundongos de 10 amostras de vírus da raiva isoladas de morcegos hematófagos e não-hematófagos, no Estado do Rio de Janeiro, sua caracterização genética quanto aos genes N e G, além da resposta de camundongos vacinados com a vacina anti-rábica produzida pela replicação da amostra Pitman-Moore em cultivo celular, frente ao desafio com estas amostras virais. As passagens sucessivas em camundongos determinaram uma redução do período de incubação e a obtenção de amostras virais com altos títulos. A vacina anti-rábica utilizada na imunização dos camundongos ofereceu proteção em mais de 80% dos camundongos vacinados com a diluição 1:5 da vacina, frente à maioria das amostras. A análise filogenética do gene da proteína N apresentou um padrão de agregação dividido em variante de morcego hematófago e variante de morcego insetívoro, com diferenças filogenéticas entre as variantes de morcego hematófago isoladas na Região Noroeste do Estado do Rio de Janeiro e aquelas isoladas nas Regiões Metropolitana e Sul do Estado. A substituição do resíduo ácido aspártico por ácido glutâmico na posição 118, encontradas na caracterização genética da proteína G das amostras 704/97 e 151/98, permite inferir que esta posição esteja relacionada à antigenicidade viral. Não foram observadas diferenças genéticas temporais entre as amostras estudadas. / In the present study we analyzed the biological behavior in mice of 10 rabies virus samples isolated from haematophagous and non-haematophagous bats in Rio de Janeiro State, its genetic characterization from genes N and G, and we also studied the response of mice vaccinated with cell culture rabies vaccine, produced with the Pitman-Moore strain, after viral challenge with bat rabies virus samples. Viral passages in mice resulted in reduced incubation period and high virus titrers. The vaccine used conferred protection in more than 80% of the mice vaccinated with 1:15 vaccine dilution, after viral challenge. N gene genetic analysis divided the samples in haematophagous bat strains and insectivorous bat strains, with phylogenetic differences between samples isolated in Northeast Region and those isolated in the Metropolitan and South Regions of Rio de Janeiro State. The substitution of an aspartic acid to a glutamic acid found in the position 118 of G gene genetic characterization from samples 704/97 and 151/98 seems to be related to viral antigenicity. There were no time-related genetic differences between the studied samples.

Rabies, one of the oldest recognized viral zoonotic diseases, is a fatal encephalomyelitis transmitted to man via contact with infected animals. Evan today, rabies still is a disease of public health concern with many potentially preventable deaths occurring mainly in Asia, Africa and Latin America. Rabies and rabies-related viruses are members of the Lyssavirus genus, which comprises the rabies virus (genotype 1), Lagos bat virus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), European bat lyssaviruses 1 and 2 (genotypes 5 and 6) and the Australian bat lyssavirus (genotype 7). Antigenic and genetic studies have shown that rabies virus strains circulating in particular host species tend to undergo genetic adaptation and evolve into distinct biotypes that differ in antigenicity and pathogenicity. Two biotypes of rabies virus are recognized in southern Africa. The first called the canid viruses, infect carnivores of the family Canidae (dogs, jackals and bat-eared foxes) and the second, the viverrid viruses, infect carnivores of the family Herpestidae (the yellow mongoose Cynictis penicil!ata and the slender mongoose Galerella sanguinea). In an endeavour to better understand the molecular epidemiology of lyssaviruses in Zimbabwe and South Africa, we analysed nucleotide sequences of the glycoprotein and the G-L intergenic region (rabies viruses) and the nucleoprotein gene (Mokola viruses). The main aim of the studies described in this thesis was to characterise lyssaviruses (genotypes I and 3) from Zimbabwe and compare them to those present in South Africa. In addition, we wanted to establish the role of the various rabies variants in rabies epizootics in the southern African subcontinent. It could be shown from this study that all the southern African canid viruses were closely related, with no general distinction between viruses from any of the canid species. Despite the general overall similarity between the canid viruses, certain phylogenetic groupings were apparent and by association with host species, geography and year of isolation, certain groups could be identified as particular epidemiological cycles. A high genetic diversity was evident amongst viverrid rabies viruses, the opposite of our observation for canid viruses. The viverrid virus groups corresponded to geographical pockets that were independent of host species. Mokola viruses from Zimbabwe were shown to be different from those from South Africa and phylogenetic relationships of these viruses were related to their geographical location of origin. This study has demonstrated the value of multinational surveillance and investigation in understanding the epidemiology of lyssaviruses in southern Africa and elsewhere in Africa. The results presented here will serve as basis for future studies on lyssaviruses in Africa and will contribute to the improved surveillance and control programs of rabies and Mokola viruses in the region. / Thesis (PhD (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Procedeu-se ao estudo comparativo entre as provas de soroneutralização (SN) e imunofluorescência indireta (IFI) para a avaliação de anticorpos anti-rábicos em soros de cães. A prova de IFI apresentou associação significante de alta ntensidade e elevada concordância com a prova de SN, não se verificando, porém, correlação significante entre os resultados obtidos por ambas as provas. A avaliação do comportamento da vacina anti-rábica, preparada em cérebros de camundongos lactentes e aplicada em cães nunca antes vacinados, revelou pela prova de SN uma ascensão lenta e de pequena magnitude e um declínio rápido, da curva das medianas dos títulos de anticorpos anti-rábicos, já aos 45 dias da vacinação. Relativamente à prova de IFI, a resposta imunitária revelou-se mais intensa, e o declínio da curva das medianas dos títulos de anticorpos ocorreu aos 60 dias da vacinação. / A comparative study was made between the serum-neutralization (SN) and indirect immunofluorescence CIIF) techniques for the evaluation of rabies antibodies in sera from dogs. The IIF technic showed significant association of high intensity and concordance with the SN technic. No significant correlation, however, was observed between the results obtained from both techniques. The evaluation of the suckling-mouse-brain rabies vaccine, applied in dogs never vaccinated before, showed by the SN technic a slow rise of small magnitude and a fast decline of the curve of the median rabies antibodies titers, as early as 4S days after vaccination. With regard to the IIF technic, the immunitary reaction showed itself more sensitive, and the decline of the curve of the median antibodies titers occurred 60 days after vaccination.

Network dynamics are thought to be the substrate of brain information processing and of mental representations. Moreover, network-wide dysfunctions are recognized to be at the core of several psychiatric and neurodegenerative disorders. Yet, our ability to target specific networks for functional or genetic manipulations remains limited. The development of monosynaptically-restricted Rabies virus, G-deleted Rabies virus (ΔG-Rabies), has greatly facilitated the anatomical investigation of neural circuits, revealing the network synaptic structure upstream of defined neuronal populations. However, the inherent cytotoxicity of the Rabies virus largely restrains its use to the mere structural characterisation of neural networks. To overcome this limitation, I generated novel tools that allow the manipulation of neural networks for the entire life of the animal, without affecting neuronal and circuit properties. I first developed a viral system obtained by engineering the Rabies virus genome to eliminate its cytotoxicity. This led to the generation of a Self-inactivating Rabies virus (SiR) that transcriptionally disappears from the infected neurons while leaving permanent genetic access to the traced network. I showed that SiR provides a virtually unlimited temporal window for the functional manipulation of neural circuits in vivo without adverse effects on neuronal physiology. To further expand our ways of intervening on neural networks function I then developed a completely virus-free system, named Genetically-Encoded TransSynaptic Shuttle (GETSS), which is the only specific genetically-encoded transsynaptic tracer to date. In this thesis, I established novel approaches that provide, for the first time, the functional and genetic access to traced network elements in vivo for the lifetime of the animal, with no cytotoxic effects, no changes in the electrophysiological properties of the traced neurons and no adverse effects on network function. This opens new horizons in the functional investigation of neural circuits and potentially represent the first approaches to experimentally monitor neural circuit remodelling in vivo.

Procedeu-se ao estudo comparativo entre as provas de soroneutralização (SN) e imunofluorescência indireta (IFI) para a avaliação de anticorpos anti-rábicos em soros de cães. A prova de IFI apresentou associação significante de alta ntensidade e elevada concordância com a prova de SN, não se verificando, porém, correlação significante entre os resultados obtidos por ambas as provas. A avaliação do comportamento da vacina anti-rábica, preparada em cérebros de camundongos lactentes e aplicada em cães nunca antes vacinados, revelou pela prova de SN uma ascensão lenta e de pequena magnitude e um declínio rápido, da curva das medianas dos títulos de anticorpos anti-rábicos, já aos 45 dias da vacinação. Relativamente à prova de IFI, a resposta imunitária revelou-se mais intensa, e o declínio da curva das medianas dos títulos de anticorpos ocorreu aos 60 dias da vacinação. / A comparative study was made between the serum-neutralization (SN) and indirect immunofluorescence CIIF) techniques for the evaluation of rabies antibodies in sera from dogs. The IIF technic showed significant association of high intensity and concordance with the SN technic. No significant correlation, however, was observed between the results obtained from both techniques. The evaluation of the suckling-mouse-brain rabies vaccine, applied in dogs never vaccinated before, showed by the SN technic a slow rise of small magnitude and a fast decline of the curve of the median rabies antibodies titers, as early as 4S days after vaccination. With regard to the IIF technic, the immunitary reaction showed itself more sensitive, and the decline of the curve of the median antibodies titers occurred 60 days after vaccination.