<p>HDV antigenomic RNA is processed in two distinct pathways; it can be cleaved at the polyA site and polyadenylated to become mRNA for the delta antigens, or the RNA can be cleaved by the antigenomic ribozyme to become full-length antigenomic RNA that is used for synthesis of genomic HDV RNA. The polyA site is located just 33 nucleotides upstream of the ribozyme cleavage site. If processing occurs primarily at the upstream polyA site, there may not be enough full-length antigenomic RNA to support replication. On the other hand, ribozyme cleavage downstream of the polyA site could inhibit polyadenylation by interfering with polyadenylation complex assembly. Thus, it appears that HDV may need a mechanism to control RNA processing so that both products can be generated in the proper amounts during the infection cycle. </p><p>A model has been proposed in which the choice between ribozyme cleavage and polyadenylation is determined by alternative RNA secondary structures formed by the polyA sequence (Wadkins and Been 2002). One of the hypothetical structures, AltP2, is a pairing between part of the upstream polyA sequence and the 3' end of the ribozyme sequence. For this model, the same upstream sequence that forms AltP2 could also form a stem loop, P(-1), within the leader, by pairing with sequences located farther upstream. A processing choice is possible because AltP2 is predicted to inhibit ribozyme cleavage and favor polyadenylation resulting in mRNA production, whereas P(-1) would inhibit polyadenylation and favor ribozyme cleavage resulting in full-length replication product. </p><p>The P(-1) vs. AltP2 model was tested using an antigenomic HDV ribozyme construct with the 60-nucleotide sequence upstream of the ribozyme cleavage site. This leader sequence contains the proposed polyA sequence elements. In vitro analysis of this construct revealed that the kinetic profile of ribozyme self-cleavage was altered in two ways. Relative to the ribozyme without upstream sequences, the fraction of precursor RNA that cleaved decreased to about 50%, but the active ribozyme fraction cleaved faster. Native gel electrophoresis revealed that the active and inactive precursor RNAs adopted persistent alternative structures, and structure mapping with Ribonuclease T1 and RNase H provided evidence for structures resembling P(-1) and AltP2.</p><p>Sequence changes in the 5' leader designed to alter the relative stability of P(-1) and AltP2 increased or decreased the extent of ribozyme cleavage in a predictable way, but disrupting AltP2 did not completely restore ribozyme activity. The analysis of deletion and base change variants supported a second alternative pairing, AltP4, formed by the pyrimidine-rich sequence immediately 5' of the ribozyme cleavage site and a purine-rich sequence from the 5' side of P4. A similar approach was used to test if the effect of disrupting both AltP2 and AltP4 might be additive, and the results suggested that ribozyme precursors with 5' leader sequences could fold into multiple inactive conformations, which can include, but may not be limited to, AltP2, AltP4, or a combination of both.</p><p>Luciferase expression constructs with HDV polyA and ribozyme sequences were used to investigate the effects of RNA structure and ribozyme cleavage on polyadenylation in cells. One hypothesis was that P(-1) could inhibit polyadenylation by making the polyA sequence elements less accessible to polyA factors, but sequence changes designed to alter the stability of the stem loop had no effect on polyadenylation. The model also predicts that the ribozyme sequence downstream of the polyA site could affect polyadenylation, possibly in two different ways. Ribozyme cleavage could interfere with polyadenylation by uncoupling transcription from processing, however, the ribozyme sequence might also influence polyadenylation in a manner independent of the ribozyme cleavage activity. As such, the AltP2 structure could potentially have a positive effect on polyadenylation either by inhibiting ribozyme cleavage or by making the polyA signal sequences more accessible to the polyA factors. To distinguish between the effects of ribozyme cleavage and alternative RNA structures, luciferase expression levels from constructs with an HDV polyA sequence followed by the active wild-type ribozyme or the inactive C76u version of the ribozyme were compared. For the wild-type HDV polyA sequence, the active ribozyme reduced expression, whereas the inactive ribozyme control had no effect on expression. However, for the modified leader sequences, which were efficiently polyadenylated in the absence of ribozyme, there were changes in expression that appeared to be independent of ribozyme cleavage. Based on these findings, two alternative models are proposed. One model predicts that protein factors might affect antigenomic RNA processing, and the other model suggests that additional alternative structures, such as AltP4, might influence the choice between ribozyme cleavage and polyadenylation.</p> / Dissertation

As hepatites virais estão entre as mais importantes pandemias mundiais da atualidade. Existem várias causas de hepatite, entre elas, o vírus da hepatite B (HBV), o vírus da hepatite C (HCV) e o vírus da Hepatite Delta (HDV). Da mesma forma, o vírus GB-C (GBV-C) é importante na coinfecção com outros vírus, como o HIV. Nesse estudo, várias regiões da América do Sul foram analisadas. Na Colômbia, os estados do Amazonas e Magdalena foram encontradas como regiões hiperendêmicas para HBV. O genótipo F3 (75%) foi o mais prevalente. Determinou-se que o subgenótipo F3 é o mais antigo dos subgenótipos F. No estado de Chocó, encontrou-se o subgenótipo A1 (52,1%) como o mais prevalente. Surpreendentemente, nesse mesmo estado foram encontrados nove casos autóctones de infecção pelo genótipo E (39,1%). Para o HCV, em Bogotá, encontrou-se o subtipo 1b (82,8%) como o mais prevalente. Da mesma forma, estimou-se que esse subtipo foi introduzido por volta de 1950 e se propagou exponencialmente entre 1970 a 1990. O HDV foi identificado em casos de hepatite fulminante do estado de Amazonas, todos classificados como genótipo 3. Se determinou que o HDV/3 se espalhou exponencialmente a partir de 1950 a 1970 na América do Sul e depois desta época, esta infecção deixou de aumentar, provavelmente devido a introdução de vacinação contra o HBV. GBV-C foi procurado em doadores de sangue colombianos infectados com HCV e/ou HBV de Bogotá e em povos indígenas com infecção pelo HBV no Amazonas. A análise filogenética revelou a presença do genótipo 2a como o mais prevalente entre os doadores de sangue e o 3 nos povos indígenas estudados. A presença do genótipo 3 na população indígena foi previamente relatada na região de Santa Marta, na Colômbia e nos povos indígenas da Venezuela e da Bolívia. No Chile, foi realizado um estudo com 21 pacientes cronicamente infectados pelo HBV sem tratamento antiviral prévio. Todas as sequências obtidas eram do subgenótipo F1b e se agrupavam em quatro diferentes grupos, sugerindo que diferentes linhagens desse subgenótipo estão circulando no Chile. No Brasil, no estado de Rondônia, para o HCV, encontramos o subtipo 1b (50,0%) como o mais frequente. Esse foi o primeiro relato sobre os genótipos do HCV neste estado. Para o HBV, o subgenótipo A1 (37,0%) foi o mais frequente. Os resultados do estado de Rondônia são consistentes com outros estudos no Brasil, mostrando a presença de vários genótipos do HBV, refletindo a origem mista da população Brasileira. Estudando o estado do Maranhão, avaliamos a frequência da infecção pelo HBV e seus genótipos. Foram encontradas 4 sequencias genótipo A1 que agruparam com outras sequências reportadas do Brasil. Em outro estudo, caracterizamos os subgenótipos do HBV em 68 pacientes com hepatite crônica B em Pernambuco, encontrando 78,7% de presença do subgenótipo A1. Finalmente, em um estudo realizado com amostras da cidade de São Paulo, encontramos um caso de HBV genótipo C em um brasileiro nativo, sendo essa a primeira sequência completa do genoma de HBV/C2 notificados no Brasil / Viral hepatitis are among the major pandemics in the world nowadays. There are many causes of hepatitis, including hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis delta virus (HDV). Similarly, GB virus C (GBV-C) is a relevant agent in co-infection with HIV. In this study, several regions of South America were studied. In Colombia, the states of Amazonas and Magdalena were identified as highly endemic areas for HBV. Genotype F3 (75%) was the most prevalent. It was determined that subgenotype F3 is the oldest among all F subgenotypes. In the state of Chocó, subgenotype A1 (52.1%) was the most prevalent. Surprisingly, nine indigenous cases of infection by genotype E (39.1%) were found in this state. For HCV, in Bogotá, subtype 1b (82.8%) was the most frequent. Likewise, it was estimated that this subtype was introduced around 1950 and spread exponentially from 1970 to 1990. HDV has been identified in cases of fulminant hepatitis in the state of Amazonas, all of them classified as genotype 3. It was determined that the HDV/3 spread exponentially from 1950 to 1970 in South America and after this time, this infection stopped to increase, probably due to introduction of vaccination against HBV. GBV-C was sought in Colombian blood donors infected with HCV and/or HBV in Bogotá and indigenous peoples with HBV infection in the Amazon. The phylogenetic analysis revealed the presence of genotype 3 as the most prevalent among blood donors and in three studied indigenous people. The presence of genotype 3 in the indigenous population has been previously reported in the region of Santa Marta, Colombia, and in the indigenous peoples of Venezuela and Bolivia. In Chile, a study was carried out with 21 patients chronically infected with HBV without any prior antiviral treatment. All sequences obtained belonged to subgenotype F1b and clustered into four different groups, suggesting that different strains that are circulating in Chile. In Brazil, the state of Rondônia, we found HCV subtype 1b (50.0%) as the most frequent. This was the first report on HCV genotypes in this state. For HBV, subgenotype A1 (37.0%) was the most frequent. The results of the state of Rondônia are consistent with other studies carried out in Brazil, showing the presence of several HBV genotypes, reflecting the mixed origin of the Brazilian population. Studying the state of Maranhão, we evaluated the frequency of HBV infection and its genotypes and we found 4 genotype A1 sequences that grouped with other sequences reported in Brazil. In another study, we characterized HBV subgenotypes in 68 patients with chronic hepatitis B in Pernambuco and we found subgenotype A1 in 78.7% cases. Finally, in a study of samples from São Paulo, we found a case of HBV genotype C in a native Brazilian patient and this is the first complete genome sequence of HBV/C2 reported in Brazil

As hepatites virais estão entre as mais importantes pandemias mundiais da atualidade. Existem várias causas de hepatite, entre elas, o vírus da hepatite B (HBV), o vírus da hepatite C (HCV) e o vírus da Hepatite Delta (HDV). Da mesma forma, o vírus GB-C (GBV-C) é importante na coinfecção com outros vírus, como o HIV. Nesse estudo, várias regiões da América do Sul foram analisadas. Na Colômbia, os estados do Amazonas e Magdalena foram encontradas como regiões hiperendêmicas para HBV. O genótipo F3 (75%) foi o mais prevalente. Determinou-se que o subgenótipo F3 é o mais antigo dos subgenótipos F. No estado de Chocó, encontrou-se o subgenótipo A1 (52,1%) como o mais prevalente. Surpreendentemente, nesse mesmo estado foram encontrados nove casos autóctones de infecção pelo genótipo E (39,1%). Para o HCV, em Bogotá, encontrou-se o subtipo 1b (82,8%) como o mais prevalente. Da mesma forma, estimou-se que esse subtipo foi introduzido por volta de 1950 e se propagou exponencialmente entre 1970 a 1990. O HDV foi identificado em casos de hepatite fulminante do estado de Amazonas, todos classificados como genótipo 3. Se determinou que o HDV/3 se espalhou exponencialmente a partir de 1950 a 1970 na América do Sul e depois desta época, esta infecção deixou de aumentar, provavelmente devido a introdução de vacinação contra o HBV. GBV-C foi procurado em doadores de sangue colombianos infectados com HCV e/ou HBV de Bogotá e em povos indígenas com infecção pelo HBV no Amazonas. A análise filogenética revelou a presença do genótipo 2a como o mais prevalente entre os doadores de sangue e o 3 nos povos indígenas estudados. A presença do genótipo 3 na população indígena foi previamente relatada na região de Santa Marta, na Colômbia e nos povos indígenas da Venezuela e da Bolívia. No Chile, foi realizado um estudo com 21 pacientes cronicamente infectados pelo HBV sem tratamento antiviral prévio. Todas as sequências obtidas eram do subgenótipo F1b e se agrupavam em quatro diferentes grupos, sugerindo que diferentes linhagens desse subgenótipo estão circulando no Chile. No Brasil, no estado de Rondônia, para o HCV, encontramos o subtipo 1b (50,0%) como o mais frequente. Esse foi o primeiro relato sobre os genótipos do HCV neste estado. Para o HBV, o subgenótipo A1 (37,0%) foi o mais frequente. Os resultados do estado de Rondônia são consistentes com outros estudos no Brasil, mostrando a presença de vários genótipos do HBV, refletindo a origem mista da população Brasileira. Estudando o estado do Maranhão, avaliamos a frequência da infecção pelo HBV e seus genótipos. Foram encontradas 4 sequencias genótipo A1 que agruparam com outras sequências reportadas do Brasil. Em outro estudo, caracterizamos os subgenótipos do HBV em 68 pacientes com hepatite crônica B em Pernambuco, encontrando 78,7% de presença do subgenótipo A1. Finalmente, em um estudo realizado com amostras da cidade de São Paulo, encontramos um caso de HBV genótipo C em um brasileiro nativo, sendo essa a primeira sequência completa do genoma de HBV/C2 notificados no Brasil / Viral hepatitis are among the major pandemics in the world nowadays. There are many causes of hepatitis, including hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis delta virus (HDV). Similarly, GB virus C (GBV-C) is a relevant agent in co-infection with HIV. In this study, several regions of South America were studied. In Colombia, the states of Amazonas and Magdalena were identified as highly endemic areas for HBV. Genotype F3 (75%) was the most prevalent. It was determined that subgenotype F3 is the oldest among all F subgenotypes. In the state of Chocó, subgenotype A1 (52.1%) was the most prevalent. Surprisingly, nine indigenous cases of infection by genotype E (39.1%) were found in this state. For HCV, in Bogotá, subtype 1b (82.8%) was the most frequent. Likewise, it was estimated that this subtype was introduced around 1950 and spread exponentially from 1970 to 1990. HDV has been identified in cases of fulminant hepatitis in the state of Amazonas, all of them classified as genotype 3. It was determined that the HDV/3 spread exponentially from 1950 to 1970 in South America and after this time, this infection stopped to increase, probably due to introduction of vaccination against HBV. GBV-C was sought in Colombian blood donors infected with HCV and/or HBV in Bogotá and indigenous peoples with HBV infection in the Amazon. The phylogenetic analysis revealed the presence of genotype 3 as the most prevalent among blood donors and in three studied indigenous people. The presence of genotype 3 in the indigenous population has been previously reported in the region of Santa Marta, Colombia, and in the indigenous peoples of Venezuela and Bolivia. In Chile, a study was carried out with 21 patients chronically infected with HBV without any prior antiviral treatment. All sequences obtained belonged to subgenotype F1b and clustered into four different groups, suggesting that different strains that are circulating in Chile. In Brazil, the state of Rondônia, we found HCV subtype 1b (50.0%) as the most frequent. This was the first report on HCV genotypes in this state. For HBV, subgenotype A1 (37.0%) was the most frequent. The results of the state of Rondônia are consistent with other studies carried out in Brazil, showing the presence of several HBV genotypes, reflecting the mixed origin of the Brazilian population. Studying the state of Maranhão, we evaluated the frequency of HBV infection and its genotypes and we found 4 genotype A1 sequences that grouped with other sequences reported in Brazil. In another study, we characterized HBV subgenotypes in 68 patients with chronic hepatitis B in Pernambuco and we found subgenotype A1 in 78.7% cases. Finally, in a study of samples from São Paulo, we found a case of HBV genotype C in a native Brazilian patient and this is the first complete genome sequence of HBV/C2 reported in Brazil