Orientador: Renate Krause Sakate / Banca: Carlos Frederico Wilcken / Banca: Arlete Marchi Tavares de Melo / Banca: Marcelo Agenor Pavan / Banca: Antonio Carlos Maringoni / Resumo: Considerando o aumento de begomovírus e mosca-branca no campo o presente trabalho teve como objetivos a detecção, a caracterização molecular e a análise da diversidade genética de begomovírus em pimentão e tomateiro em diferentes municípios do Estado de São Paulo: Piraju, Tejupá, Santa Cruz do Rio Pardo, São Pedro do Turvo, São Miguel Arcanjo, Itapetininga, Lins, Sabino, Timburí, Iacanga, Pirajuí, Avaí, Reginópolis e Salto; a identificação de biótipos de B. tabaci por meio da amplificação do gene mitocondrial (citocromo oxidase I - mtCOI) seguido de seqüenciamento ou RFLP utilizando a enzima Taq I e a avaliação para resistência de acessos de Capsicum spp. a dois isolados de ToSRV. A análise da variabilidade foi realizada por meio de seqüenciamento da região da capa protéica (DNA-A) com oligonucleotídeos universais e, paralelamente, as mesmas amostras foram testadas por amplificação por círculo rolante (RCA) sendo, posteriormente, clivadas com a enzima de restrição HpaII. Um total de 812 amostras foi analisado, sendo 709 de pimentão e 103 de tomate. Por PCR tradicional, foram detectadas positivas para presença de begomovírus 98 amostras provenientes de pimentão e 39 de tomateiro, e por RCA-PCR, foram 332 e 82 respectivamente, evidenciando maior sensibilidade desta técnica. Dessas amostras, foram seqüenciadas 39 de pimentão e 25 de tomateiro, verificando-se ocorrência prevalente da espécie ToSRV no estado de São Paulo. Infecção mista com ToSRV e ToYVSV foi observada tomateiro. Por RCA-RFLP, foram observados quatro padrões de clivagem com a enzima HpaII e todos foram confirmados como sendo da espécie ToSRV indicando variabilidade molecular intraespecífica. Para tomateiro, foram observados 18 padrões de restrição, dois idênticos aos verificados em plantas de pimentão indicando, possivelmente, infecção pelos mesmos isolados de ToSRV, porém... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Considering the high incidence of begomoviruses and the whitefly on the field, the objetives of this work were to analyze the genetic diversity of begomoviruses infecting pepper and tomato plants in different counties of São Paulo State: Piraju, Tejupá, Santa Cruz do Rio Pardo, São Pedro do Turvo, São Miguel Arcanjo, Itapetininga, Lins, Sabino, Timburí, Iacanga, Pirajuí, Avaí, Reginópolis and Salto; the identification of biotypes of B. tabaci through the amplification of the mitochondrial gene (cytochrome oxidase I) 4 followed by sequencing the gene or analysis by RFLP using the enzyme TaqI and the evaluation of Capsicum spp. for the resistance source for two isolates of ToSRV. The coat protein from the DNA A of the begomovirus was amplified and sequenced, and the same samples were amplified by rolling circle amplification (RCA) followed by analysis by RCA-RFLP using the HpaII enzyme. A total of 812 samples were analyzed, 709 from pepper and 103 from tomato. By PCR, 98 samples from pepper and 39 from tomato were positives for the presence of begomoviruses, while by RCA-PCR 332 and 82 respectively. Thirty-nine samples from pepper and 25 from tomato were sequenced indicating the prevalence of the ToSRV species in São Paulo State. Mixed infections with ToSRV and ToYVSV were found in tomato plants. By RCA-RFLP four restriction profiles were found for ToSRV in pepper plants. In tomato 18 profiles were observed: two identical as observed for ToSRV in pepper, indicating possible infection with the same ToSRV isolates, a profile for ToSRV and ToYVSV mixed infections and also different profiles for ToSRV isolates didn't found in pepper plants. The sequencing of 17 samples of B. tabaci mitochondrial citochrome oxidase I gene and analysis by Taq I digestion of whiteflies collected in growers areas of pepper and tomato indicated only the presence of the B biotype... (Complete abstract click electronic access below) / Doutor

A utilização ampla do glyphosate como herbicida na agricultura brasileira e mundial é conseqüência, dentre outros fatores, de seu custo relativamente baixo, alta eficácia no controle de plantas daninhas, amplo espectro de controle de espécies de plantas daninhas, baixa toxicidade, e curta persistência no ambiente. No entanto, o uso intensivo do glyphosate tem proporcionado a seleção de biótipos de plantas daninhas resistentes a este herbicida, como é o caso da buva (Conyza bonariensis). Apesar de estudos já terem sido conduzidos pela comunidade científica de diversos países, para elucidar o fenômeno sob diversos aspectos, no Brasil são poucos os estudos científicos que esclareçam os mecanismos de resistência de plantas daninhas ao glyphosate. Neste sentido, esta pesquisa teve por objetivo estudar comparativamente populações resistentes (R) e suscetíveis (S) de C. bonariensis ao herbicida glyphosate para caracterizar o nível de resistência das populações R; comparar os níveis de acúmulo de ácido shiquímico entre as populações R e S; elucidar os principais mecanismos de resistência dos biótipos R, através da análise da absorção e translocação do herbicida, determinar a expressão gênica da EPSPS, através de análises semi-quantitativas RTPCR e sugerir alternativas químicas de controle para buva em pomares cítricos do Estado de São Paulo. Os resultados obtidos através do estudo de ácido shiquímico indicaram que o mecanismo de resistência das populações R buva estudadas não está relacionado com insensibilidade da EPSPS ao glyphosate. Já os estudos de translocação diferencial evidenciaram ser esta uma das causas dos mecanismos envolvidos na resistência do biótipo R. Através da análise da expressão gênica observou-se alto grau de similaridade entre seqüências obtidas dos genes EPSPS de C. bonariensis e as seqüências de C. canadensis depositadas no GenBank. Além disso, é possível sugerir que há relação entre a expressão dos genes EPSPS em C. bonariensis e a condição de resistência à ação do herbicida glyphosate em alguns indivíduos dessa espécie de planta daninha. / The extensive use of glyphosate as herbicide in the Brazilian and worldwide agriculture is a consequence, among other factors, of the relatively low cost, high weed control efficacy; wide weed species control spectrum; low toxicity, and short persistence in the environment. However, the intensive use of glyphosate has imposed the selection of certain resistant weed biotypes to this herbicide, such as the case of the weed Conyza bonariensis. Despite the fact that some studies has been developed in some countries around the world, in order to elucidate the phenomenon in several aspects, in Brazil a few studies have been conducted scientifically in order to elucidate the mechanisms of weed resistance to glyphosate. Therefore, this research was developed with the objective of studying the populations of C. bonariensis to characterize the resistance level of populations to glyphosate; compare the levels of shikimic acid accumulation between resistant (R) and susceptible (S) plants; elucidate the main mechanisms of resistance of resistant biotypes to glyphosate, by the absorption and translocation of the herbicide, and determine the EPSPS gene expression, by the RT-PCR semi-quantitative analysis. The results obtained by the shikimic acid study indicated that the mechanism of resistance of the biotype of C. bonariensis studied is not related with insensitivity of the EPSPS to glyphosate. On the other hand, the differential translocation maybe considered as one of the mechanisms involved in the resistance of the biotype R of C. bonariensis. With regards to gene expression, it was observed high degree of similarity among the sequences obtained of the EPSPS gene of C. canadensis, and the sequences of C. canadensis deposited in the GenBank. Furthermore, it is possible to suggest that there is relation between the gene expression in C. bonariensis and the resistance condition to the action of the herbicide glyphosate in some individuals of this weed specie.

O fungo Moniliophthora perniciosa é o agente causal da vassoura de bruxa no cacaueiro (Theobroma cacao L.). Três biótipos distintos (biótipos -C, -L e -S) são reconhecidos de acordo com a especificidade quanto ao hospedeiro. O presente estudo teve como objetivo a análise da diversidade genética com o uso de marcadores microssatélites de 134 isolados dos biótipos -C, -L e -S de M. perniciosa coletados principalmente na Amazônia Brasileira e áreas de cultivo. A diversidade genética foi associada com virulência e/ou agressividade dos isolados a acessos diferenciais (suscetíveis ou resistentes) de T. cacao. Os biótipos -S e -L apresentaram uma diversidade gênica e genotípica superior em comparação com o biótipo-C. A população do biótipo-C com maior diversidade genotípica foi a do Acre, seguida pelo Oeste do Amazonas, ambas correspondendo a áreas em que se encontra cacaueiro nativo no Brasil. A população com menor diversidade genotípica foi a da Bahia, que corresponde a uma área onde a presença de M. perniciosa foi registrada mais recentemente, no final da década de 1980. Dos 134 isolados, 83 correspondem a genótipos multilocos únicos, sendo encontrados apenas dois indivíduos com genótipos idênticos para o biótipo-S, e nenhum para o biótipo-L. No biótipo-C foram identificados 61 genótipos multilocos em 111 isolados coletados em áreas de ocorrência natural e cultivo de cacau. Os dados de similaridade genética corroboram que o biótipo-C e também o -S que são homotálicos evoluíram de um biótipo heterotálico, possivelmente o biótipo-L. As populações do biótipo-C do estado do Pará e Leste do Amazonas compartilham ancestrais comuns em Ji-Paraná (RO) e Assis Brasil (AC), enquanto que a região sob a influência do rio Amazonas possui outra ascendência, que seria em Atalaia do Norte (Alto Solimões). Os genótipos multilocos da Bahia exibiram origens análogas as da população do Baixo Amazonas, com ascendência em Ji-Paraná (RO) e Alenquer (PA). Na avaliação de agressividade de M. perniciosa, a concentração do inóculo se mostrou determinante para a manifestação dos sintomas, com o aumento de plântulas com sintomas à medida que aumenta a concentração de basidiósporos. Dentre as progênies avaliadas, \'PA 195 x CAB 214\' apresentou menor proporção de plântulas com sintomas. Na inoculação com isolados de M. perniciosa oriundos do Acre e Amazonas, a proporção de plântulas com sintomas foi superior quando inoculadas com o isolado de Tabatinga (AM). O genótipo de cacaueiro que apresentou uma reação diferenciada foi o \'CAB 214\', para o qual nenhuma plântula apresentou sintomas quando inoculada com o isolado de Marechal Thaumaturgo (AC). Com o uso de microssatélites os genótipos multilocos de Tabatinga (AM) e Marechal Thaumaturgo (AC) foram identificados em grupos distintos. Na análise de treze genes de patogenicidade induzidos pela limitada disponibilidade de N, o gene 88KD foi o que demonstrou o maior número de transcritos acumulados. Os isolados que apresentaram uma mesma tendência em seus genes mais expressos foram Tabatinga (AM) e Óbidos (PA) que apesar de possuírem origens geográficas distintas, foram identificados no mesmo grupo na análise com microssatélites / The fungus Moniliophthora perniciosa is the causal agent of witches\'broom in cacao (Theobroma cacao L.). Three different biotypes (C-; S-; and L-biotypes) are recognized according to host specificity. The present study aimed to analyze the genetic diversity using microsatellite markers for 134 isolates of the C-, L- and S- biotypes of M. perniciosa collected mainly in the Brazilian Amazon and areas of cultivation. Genetic diversity was associated with virulence and/or aggressiveness of isolates in differential accesses (susceptible or resistant) of T. cacao. The L- and S- biotypes showed a higher genetic and genotype diversity compared with C-biotype. Of the 134 isolates, 83 corresponded to unique multilocus genotypes, and found only two individuals with identical genotypes for the S-biotype, and none for the L-biotype. In the C-biotype were identified 61 multilocus genotypes in 111 isolates collected in areas of natural occurrence and cultivation of cocoa. The genetic similarity data corroborate that the C- and S- biotypes that are apparently homothallic evolved from a heterothallic biotype, possibly L-biotype. The populations of C-biotype of the state of Para and Amazonas East share common ancestors, in Ji-Paraná (RO) and Assis Brazil (AC), while the region under the influence of the Amazon River has another descent that would be in the Atalaia do Norte (Upper Solimões). The multilocus genotypes from Bahia showed a similar origin of the population of the Lower Amazon, with ancestry in Ji-Paraná (RO) and Alenquer (PA). In the evaluation of aggressiveness of M. perniciosa, the inoculum concentration proved to be decisive for the manifestation of symptoms, with the increase of seedlings with symptoms as increases the concentration of basidiospores. Among the progenies, \'PA 195 x CAB 214\' showed a lower proportion of seedlings with symptoms. In inoculation with M. perniciosa from Acre and Amazonas, the proportion of seedlings with symptoms was higher when inoculated with the isolate from Tabatinga (AM). The genotype of cocoa that had a differentiated reaction was the \'CAB 214\', for which no plants showed symptoms when inoculated with the isolate Marechal Thaumaturgo (AC). With the use of microsatellite, the multilocus genotypes of Tabatinga (AM) and Marechal Thaumaturgo (AC) were identified in different groups. In the analysis of thirteen genes of pathogenicity induced by the limited availability of N, 88KD gene showed the highest number of transcripts. The isolates that showed a similar trend in their more expressed genes were Tabatinga (AM) and Óbidos (PA) that despite having different geographical origins were identified in the same group in the analysis with microsatellite

O fungo Moniliophthora perniciosa é o agente causal da vassoura de bruxa no cacaueiro (Theobroma cacao L.). Três biótipos distintos (biótipos -C, -L e -S) são reconhecidos de acordo com a especificidade quanto ao hospedeiro. O presente estudo teve como objetivo a análise da diversidade genética com o uso de marcadores microssatélites de 134 isolados dos biótipos -C, -L e -S de M. perniciosa coletados principalmente na Amazônia Brasileira e áreas de cultivo. A diversidade genética foi associada com virulência e/ou agressividade dos isolados a acessos diferenciais (suscetíveis ou resistentes) de T. cacao. Os biótipos -S e -L apresentaram uma diversidade gênica e genotípica superior em comparação com o biótipo-C. A população do biótipo-C com maior diversidade genotípica foi a do Acre, seguida pelo Oeste do Amazonas, ambas correspondendo a áreas em que se encontra cacaueiro nativo no Brasil. A população com menor diversidade genotípica foi a da Bahia, que corresponde a uma área onde a presença de M. perniciosa foi registrada mais recentemente, no final da década de 1980. Dos 134 isolados, 83 correspondem a genótipos multilocos únicos, sendo encontrados apenas dois indivíduos com genótipos idênticos para o biótipo-S, e nenhum para o biótipo-L. No biótipo-C foram identificados 61 genótipos multilocos em 111 isolados coletados em áreas de ocorrência natural e cultivo de cacau. Os dados de similaridade genética corroboram que o biótipo-C e também o -S que são homotálicos evoluíram de um biótipo heterotálico, possivelmente o biótipo-L. As populações do biótipo-C do estado do Pará e Leste do Amazonas compartilham ancestrais comuns em Ji-Paraná (RO) e Assis Brasil (AC), enquanto que a região sob a influência do rio Amazonas possui outra ascendência, que seria em Atalaia do Norte (Alto Solimões). Os genótipos multilocos da Bahia exibiram origens análogas as da população do Baixo Amazonas, com ascendência em Ji-Paraná (RO) e Alenquer (PA). Na avaliação de agressividade de M. perniciosa, a concentração do inóculo se mostrou determinante para a manifestação dos sintomas, com o aumento de plântulas com sintomas à medida que aumenta a concentração de basidiósporos. Dentre as progênies avaliadas, \'PA 195 x CAB 214\' apresentou menor proporção de plântulas com sintomas. Na inoculação com isolados de M. perniciosa oriundos do Acre e Amazonas, a proporção de plântulas com sintomas foi superior quando inoculadas com o isolado de Tabatinga (AM). O genótipo de cacaueiro que apresentou uma reação diferenciada foi o \'CAB 214\', para o qual nenhuma plântula apresentou sintomas quando inoculada com o isolado de Marechal Thaumaturgo (AC). Com o uso de microssatélites os genótipos multilocos de Tabatinga (AM) e Marechal Thaumaturgo (AC) foram identificados em grupos distintos. Na análise de treze genes de patogenicidade induzidos pela limitada disponibilidade de N, o gene 88KD foi o que demonstrou o maior número de transcritos acumulados. Os isolados que apresentaram uma mesma tendência em seus genes mais expressos foram Tabatinga (AM) e Óbidos (PA) que apesar de possuírem origens geográficas distintas, foram identificados no mesmo grupo na análise com microssatélites / The fungus Moniliophthora perniciosa is the causal agent of witches\'broom in cacao (Theobroma cacao L.). Three different biotypes (C-; S-; and L-biotypes) are recognized according to host specificity. The present study aimed to analyze the genetic diversity using microsatellite markers for 134 isolates of the C-, L- and S- biotypes of M. perniciosa collected mainly in the Brazilian Amazon and areas of cultivation. Genetic diversity was associated with virulence and/or aggressiveness of isolates in differential accesses (susceptible or resistant) of T. cacao. The L- and S- biotypes showed a higher genetic and genotype diversity compared with C-biotype. Of the 134 isolates, 83 corresponded to unique multilocus genotypes, and found only two individuals with identical genotypes for the S-biotype, and none for the L-biotype. In the C-biotype were identified 61 multilocus genotypes in 111 isolates collected in areas of natural occurrence and cultivation of cocoa. The genetic similarity data corroborate that the C- and S- biotypes that are apparently homothallic evolved from a heterothallic biotype, possibly L-biotype. The populations of C-biotype of the state of Para and Amazonas East share common ancestors, in Ji-Paraná (RO) and Assis Brazil (AC), while the region under the influence of the Amazon River has another descent that would be in the Atalaia do Norte (Upper Solimões). The multilocus genotypes from Bahia showed a similar origin of the population of the Lower Amazon, with ancestry in Ji-Paraná (RO) and Alenquer (PA). In the evaluation of aggressiveness of M. perniciosa, the inoculum concentration proved to be decisive for the manifestation of symptoms, with the increase of seedlings with symptoms as increases the concentration of basidiospores. Among the progenies, \'PA 195 x CAB 214\' showed a lower proportion of seedlings with symptoms. In inoculation with M. perniciosa from Acre and Amazonas, the proportion of seedlings with symptoms was higher when inoculated with the isolate from Tabatinga (AM). The genotype of cocoa that had a differentiated reaction was the \'CAB 214\', for which no plants showed symptoms when inoculated with the isolate Marechal Thaumaturgo (AC). With the use of microsatellite, the multilocus genotypes of Tabatinga (AM) and Marechal Thaumaturgo (AC) were identified in different groups. In the analysis of thirteen genes of pathogenicity induced by the limited availability of N, 88KD gene showed the highest number of transcripts. The isolates that showed a similar trend in their more expressed genes were Tabatinga (AM) and Óbidos (PA) that despite having different geographical origins were identified in the same group in the analysis with microsatellite

Στην παρούσα μελέτη η βιοτυπία και η ανάλυση του DNA με ηλεκτρο-φόρηση σε παλλόμενο ηλεκτρικό πεδίο (PFGE) εφαρμόσθηκαν σε ένα σύνο-λο εντε-ρο-κόκ-κων που απομονώθηκαν από νερά αναψυχής και πόσιμα νερά, προκει-μένου να προσδιορισθεί πιθανή γενετική συγγένεια. Τα 200 στελέχη εντε-ρο--κόκκου απομονώθηκαν από 246 δείγματα νερών αναψυχής και 903 δείγματα πόσιμου νερού, από θάλασσα, νερό δικτύου, ποταμούς και πηγές της Ν.Δ. Ελλάδας. Η ταυτοποίηση σε επίπεδο είδους έγινε με το σύστημα API 20strep. Ο έλεγχος της ευαισθησίας στα αντιβιοτικά έγινε με προσδιορισμό της ελάχιστης ανασταλτικής πυκνότητας (MIC) με την μέθοδο ταινιών E-test. Η ηλεκτροφό-ρηση σε παλλόμενο ηλεκτρικό πεδίο εφαρμόσθηκε για τις γονοτυπικές δοκιμασίες. Ο χαρακτηρισμός σε επίπεδο είδους ταξινόμησε τους εντερόκοκκους σε 22 βιότυπους. Στην πλειοψηφία τους τα στελέχη ήσαν E. faecium (142 ή 71%), ακολουθούσε ο E. faecalis (40 ή 20%), o E. durans (9 ή 4,5%), o E. gallinarum (5 ή 2,5%) και o E. avium (4 ή 2%). Μεταξύ των στελεχών του E. faecium υπήρ-χε σχέση των βιοτύπων και της πηγής του δείγματος. Αυτό δεν παρατηρή-θη-κε στα στελέχη του E. faecalis ούτε στα υπόλοιπα είδη. Κανένα στέλεχος δεν βρέθηκε να παράγει β-λακταμάση. Δεν παρατηρήθηκε καμία σχέση μεταξύ της αντοχής στα αντιβιοτικά και της προέλευσης των στελεχών. Κανένα είδος εντεροκόκκου δεν παρουσίασε αντοχή στα γλυκοπεπτίδια. Κλινικά στελέχη E. faecium περιελήφθησαν στα γονοτυπικά πειράματα ως μάρτυρες για τους ήδη ταυτοποιημένους κλώνους που ενδημούν στο Πανεπιστημιακό Νοσοκομείο της Πάτρας. Η ανάλυση των τύπων PFGE μεταξύ των 104 στελεχών αποκάλυψε την παρουσία 18 κλώνων μεταξύ του E. faecium, 14 του E. faecalis, 2 του E. durans, 3 του E. gallinarum και ενός του E. avium. Αν και μεταξύ των εξετασθέ-ντων στελεχών παρατηρήθηκε γενετική ποικιλία, προσδιορίσθηκαν κοινοί κλώ-νοι μεταξύ διαφορετικών δειγμάτων νερών. Τα δενδρογράμματα αποκά-λυ-ψαν γενετική σχέση μεταξύ συγκεκριμέ-νων περιβαλλοντικών στελεχών του E. faecium και αυτών που ήσαν νοσοκομειακής προέλευσης. Για τη μελέτη της παρουσίας κλώνων μεταξύ εντεροκόκκων που απομονώνονται από το περιβάλλον πρέπει να εφαρμόζονται μέθοδοι ανάλυσης χρωμοσωμικού DNA. / In this study we have identified the enterococcal species isolated from different environmental sources and we have characterized their biotypes, antibiotic resistance patterns and PFGE types. Biotyping and DNA fingerprinting by pulsed-field gel electrophoresis was applied to a collection of enterococci recovered form recreational and drinking water, in order to identify possible genetic relationships. Clinical strains of hospital origin were compared to the environmental isolates. A total of 200 enterococci were isolated from 246 recreational water, and 900 drinking water. One hundred forty two isolates were characterized as Enterococcus faecium recovered from all sources, 40 E. faecalis, 9 E. durans, 5. E. gallinarum and E. avium. Biotypes, determined with API 20 strep, among E. faecium were correlated with certain environmental sources, while antibiotypes, determined with Etest, did not reveal any relationship with the sample origin. Even though genetic diversity was observed among the studied strains, common clonal types were also identified in different sources, suggesting a possible common origin of the enterococci. Cluster analysis revealed a genetic relationship between certain environmental E. faecium and clinical strains.

The lyssavirus genus of the Rhabdovirus family is divided into seven genotypes. Genotype 3, Mokola virus, has only been found on the African continent, and has been reported to infect rodents, cats, dogs and humans. The first Mokola virus identification in South Africa was made in 1970, on the east coast of the KwaZulu-Natal province. After 25 years, Mokola virus was again identified in three cats, 650 km south-west of the previous isolation. In 1997 two more Mokola infections were identified in Pinetown, only about 23 km south-west of the 1970 isolation. Phylogenetic analysis of the nucleic acid sequences of the nucleoprotein gene region of the Mokola genome, indicated that the Mokola viruses from the same geographical region were more closely related, irrespective of the time of isolation. The identification of these two distinct clusters of Mokola in South Africa leads i us to believe that this virus is more widespread than previously thought, but that the reservoir host species remains to be identified. Genotype 1 in the Rhabdovirus family, rabies virus, is found on all continents, except Australia, New Zealand, Papua New Guinea, Japan, Hawaii, Taiwan, United Kingdom, Ireland, etc. An ongoing rabies enzootic in southern Africa is associated with two genetically distinct groups of viruses, called the canid biotype (infecting carnivores of the family Canidae) and the viverrid biotype (infecting carnivores of the subfamily Viverrinae). We identified the first cases of spillover of canid biotype virus into viverrid hosts, using monoclonal antibody and nucleic acid sequence analysis. Genetic analysis of the G-L intergenic region of the rabies virus genome, showed that these spillover events do not bring about any significant change on this part of the virus genome. All of these spillover isolates maintained a typical canid virus phylogeny. Rabies viruses associated with the family Viverridae form a highly diverse group of viruses, which can be divided into four distinct phylogenetic groups, each associated with a specific geographical area in South Africa. The canid biotype of rabies virus is divided into three specific groups, based on geographic location and the associated reservoir species, namely KwaZulu-Natal province (with domestic dogs as its main vector), the western parts of South Africa (bat-eared foxes) and the northern parts of South Africa (black-backed jackals). In order to determine the degree of genetic change in the virus over a period of time, we identified two endemic canid rabies regions (KwaZulu-Natal and the northern parts of South Africa) and analysed the nucleic acid sequence variation 0f the viruses over 15 years. Phylogenetic analysis of the variable G-L intergenic region of t e virus genome indicated that the canid rabies biotype changed less than 1% over the period studied. This implies that the highly diverse viverrid biotype has been circulating in the southern African wildlife for a very long time. In order to obtain a faster, more economical, and reliable method for rabies virus biotype identification, a competitive, hemi-nested PCR assay was developed. In a single tube, two biotype specific oligonucleotides (developed by Jaftha, 1997), and a common downstream primer were -used in the biotype specific, second round amplification. The specific virus biotypes were identified on the basis of specific amplicon sizes for each biotype. A third biotype specific primer was designed to target a region of the Nucleoprotein gene, this primer was used in a second round hemi-nested reaction. Despite having been designed to specifically amplify canid biotype viruses, this primer amplified all rabies biotypes non¬specifically. We conclude that the nucleoprotein genes are too conserved to make this part of the genome a good target for a biotype-specific PCR diagnostic assay. / Dissertation (MSc (Agric) Microbiology)--University of Pretoria, 1997. / Microbiology and Plant Pathology / unrestricted