Thesis (M.Sc. in Microbiology) -- Mahidol University, 1976.

Please read the abstract in the section 00front of this document / Thesis (PhD (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

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Orientador: Jane Megid / Banca: Hélio Langoni / Banca: Marcos Bryan Heinemann / Resumo: A utilização de métodos sensíveis e específicos para o diagnóstico da raiva constitui uma importante ferramenta para o controle e profilaxia dessa enfermidade. A Reação em Cadeia pela Polimerase através de Transcriptase-Reversa (RT-PCR) tem sido utilizada com bons resultados no diagnóstico do vírus rábico, mesmo quando as amostras estão em estágio de decomposição. Adicionalmente as técnicas moleculares têm sido utilizadas para estudos epidemiológicos possibilitando um melhor conhecimento da epidemiologia viral. O presente trabalho teve como objetivo avaliar as técnicas de RT-PCR e hnRT-PCR para a detecção do vírus rábico em estudos retrospectivos, para isso foram avaliadas 101 amostras cerebrais de diferentes espécies animais armazenadas por diferentes períodos, recém-descongeladas e mantidas em temperatura ambiente por 72 horas para decomposição. Os resultados das técnicas de RT-PCR e hnRT-PCR foram comparados com resultados prévios da Imunofluorescência Direta (IFD) e Inoculação Intracerebral em Camundongos (IC). Das 50 amostras positivas testadas, 26 (52%) apresentaram resultados positivos para a RT-PCR e 45 (90%) com a associação da hnRT-PCR quando realizadas em amostras recentemente descongeladas. Das amostras em decomposição foram analisadas 48 previamente positivas; onde 17 (34,3%) apresentaram resultado positivo para a RT-PCR e 36 (75%) com a associação da hnRT-PCR. Não foram encontrados resultados falso-positivos nas amostras negativas submetidas às técnicas de biologia molecular. A hnRT-PCR apresentou maior sensibilidade em relação à RT-PCR nas amostras recém-descongeladas e em decomposição. Os resultados sugerem a viabilidade de sua aplicação em estudos retrospectivos em materiais descongelados e decompostos. / Abstract: The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerse Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and better knowledge of viral epidemiology. The aim of this work was to evaluate the RT-PCR and hnRT-PCR in rabies virus detection in retrospectives studies. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition. The RT-PCR and hnRT-PCR results were compared with preview results from Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples submitted to the molecular techniques. These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR presented greater sensibility than the standards techniques (IFD e IC) in decomposed materials for rabies diagnosis. These results suggest the viability of the application of molecular techniques in thawed and decomposed materials for retrospective studies. / Mestre

The African continent sustains a variety of lyssaviruses and this study focused on two of these lyssaviruses that are unique to the continent namely rabies virus mongoose biotype and Mokola virus (MOKV). Rabies virus (RABV) belongs to genotype (gt) 1 of the Lyssavirus genus in the family Rhabdoviridae, order Mononegavirales, while Mokola virus belongs to gt3 of this genus. Both these viruses cause fatal rabies encephalitis in vertebrate animals. Genotype 1 (rabies virus) isolates from southern African countries display great genetic diversity and are grouped into two main biotypes i.e. canid and mongoose biotypes. Due to the difference in the epidemiology and pathogenesis of these biotypes, it has been hypothesized that the two biotypes were introduced into Africa at different times. The objective was to study the molecular phylogeny of representative rabies virus isolates of the mongoose biotype, isolated in South Africa and Zimbabwe over a period of 27 years, towards a better understanding of the origin of this group. In this study the complete nucleoprotein (1353 nucleotides) and glycoprotein (1575 nucleotides) genes were sequenced. The evolutionary dynamics of this virus variant was investigated using Bayesian methodology, allowing for rate variation among the different viral lineages. The phylogenetic analysis of this dataset confirms previous findings of extended evolutionary adaptation of isolates in specific geographic areas. Furthermore when these isolates are analyzed together with rabies virus isolates from across the world, they still form an independent cluster separate from any other African rabies virus isolates, thereby hinting towards a separate introduction to the continent before that of canid rabies. Molecular clock analysis estimates the age of the mongoose biotype to be approximately 200 years old, which is in concurrence with literature describing rabies in mongooses since the early 1800’s. In addition, a phylogenetic analysis of Mokola virus isolates (gt3) from South Africa, Zimbabwe, Cameroon and Central African Republic is described. All the South African isolates before 2008, as well as most of the Zimbabwean isolates (except isolate 21846) were included in this analysis. The complete nucleoprotein gene (1353nt) was amplified and sequenced. Phylogenetic analysis showed virus grouping to correspond to their geographic location. Further analysis showed Mokola virus isolates to display genetic diversity similar to that found in representative gt1 isolates. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Canine-mediated rabies is a serious zoonosis responsible for at least 55,000 human deaths every year, primarily in less developed communities in Asia and Africa where domestic dogs are free-roaming. The disease can be effectively controlled through vaccinating at least 45% of the dogs in a population; however the impact of vaccinations on disease incidence may be affected by dynamic demographic and immunological processes. Specifically, the contribution of these processes, and their regulatory factors, to vaccination coverage and rabies transmission has not been comprehensively estimated. To improve rabies control, through field interventions and epidemiological modelling, more information regarding the effect of these processes, and their regulatory factors, on population and disease dynamics and vaccinal responses was needed. This required a multifaceted approach, using techniques from the fields of population ecology, vaccine-immunology, social science and epidemiological modelling. Demographic data were collected from four populations of free-roaming domestic dogs, two in South Africa and two in Indonesia where rabies is endemic. Longitudinal, individual-level data were obtained by direct observation and surveys, and community-level data by participatory methods. Longitudinal, serological data were collected from three cohorts within the populations. Epidemiological models were based on epidemic theory and empirical data from this current study and previous studies. A wide array of data were generated relevant to planning rabies control programmes, however of particular importance was evidence regarding positive and negative the impacts of human factors on population and disease dynamics. Nearly all of the dogs were owned, despite being free-roaming, and were accessible for vaccination through their owners; and population size was regulated through human demand for dogs and a substantial fraction of dogs was acquired from outside the communities. These translocated dogs may contribute to the spread of rabies, necessitating widespread and sustained vaccination programmes. Considerable differences in the handleability of dogs between locations and, thus ease of vaccine delivery, may also be attributable to differences in human-dog interactions. Finally, a critical review of the literature, and evaluation of epidemiological models, suggests that human interference in the transmission processes may reduce the incidence of rabies and vaccination threshold. This study has provided specific evidence that human behaviours are likely to be critically important in relation to the transmission and control of canine-mediated rabies ? and is the first to properly identify this. Further detailed studies are required to explore these behaviours and how they vary culturally and geographically. In addition, the results highlight the critical role that demographic processes more generally, as well as immunological decay, play in influencing the long term success of rabies vaccination programmes. Overall, this research has provided valuable support for planning rabies control programmes and for parameterisation of epidemiological models of infectious diseases, including rabies.

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