Caracteriza????o da infec????o pelos v??rus da hepatite B e D em plasma e tecido hep??tico de portadores cr??nicos

Submitted by Felipe Guedes Amorim (felipe_guedes17@hotmail.com) on 2018-10-22T13:50:51Z No. of bitstreams: 3 CARACTERIZA????O DA INFEC????O PELOS V??RUS DAS HEPATITES B E D EM PLASMA E TECIDO HEP??TICO DE PORTADORES CR??NICOS.pdf: 3452813 bytes, checksum: e271da19b24cd37ea8a4e33354900f20 (MD5) CARTA DE ENCAMINHAMENTO.pdf: 414411 bytes, checksum: 9dd0a540145c49dfcecf128512528fb0 (MD5) ATA DE DEFESA.jpg: 705457 bytes, checksum: f4791ecb5b56473f31626ba9bbf907e4 (MD5) Approved for entry into archive by PPGBIOTEC Biotecnologia (ppg_biotec.ufam@yahoo.com.br) on 2018-10-22T14:18:55Z (GMT) No. of bitstreams: 3 CARACTERIZA????O DA INFEC????O PELOS V??RUS DAS HEPATITES B E D EM PLASMA E TECIDO HEP??TICO DE PORTADORES CR??NICOS.pdf: 3452813 bytes, checksum: e271da19b24cd37ea8a4e33354900f20 (MD5) CARTA DE ENCAMINHAMENTO.pdf: 414411 bytes, checksum: 9dd0a540145c49dfcecf128512528fb0 (MD5) ATA DE DEFESA.jpg: 705457 bytes, checksum: f4791ecb5b56473f31626ba9bbf907e4 (MD5) Approved for entry into archive by Divis??o de Documenta????o/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-10-22T18:27:33Z (GMT) No. of bitstreams: 3 CARACTERIZA????O DA INFEC????O PELOS V??RUS DAS HEPATITES B E D EM PLASMA E TECIDO HEP??TICO DE PORTADORES CR??NICOS.pdf: 3452813 bytes, checksum: e271da19b24cd37ea8a4e33354900f20 (MD5) CARTA DE ENCAMINHAMENTO.pdf: 414411 bytes, checksum: 9dd0a540145c49dfcecf128512528fb0 (MD5) ATA DE DEFESA.jpg: 705457 bytes, checksum: f4791ecb5b56473f31626ba9bbf907e4 (MD5) Made available in DSpace on 2018-10-22T18:27:33Z (GMT). No. of bitstreams: 3 CARACTERIZA????O DA INFEC????O PELOS V??RUS DAS HEPATITES B E D EM PLASMA E TECIDO HEP??TICO DE PORTADORES CR??NICOS.pdf: 3452813 bytes, checksum: e271da19b24cd37ea8a4e33354900f20 (MD5) CARTA DE ENCAMINHAMENTO.pdf: 414411 bytes, checksum: 9dd0a540145c49dfcecf128512528fb0 (MD5) ATA DE DEFESA.jpg: 705457 bytes, checksum: f4791ecb5b56473f31626ba9bbf907e4 (MD5) Previous issue date: 2018-07-18 Funda????o de Amparo a Pesquisa do Amazonas (FAPEAM) The hepatitis B virus (HBV) is responsible for a serious global public health problem, and the problem becomes even greater when coinfection or superinfection of D virus (HDV). It is estimated that of the 240 million people worldwide with chronic HBV infection, 15 million are carriers of HDV infection. Some regions of the Amazon Basin, particularly the State of Amazonas, are considered endemic areas of infection by these viruses. HDV is classified into eight different types (HDV-1-HDV-8) and HBV in ten (A-J). Thus, this research aims to characterize the genotypes of hepatitis B and D virus in plasma and hepatic tissue and to investigate possible association of genotypes with the evolution of the disease. Plasma and hepatic tissue samples from chronic HBsAg-reactive patients coinfected with HDV were analyzed. Plasma VHD viral load was quantified by real-time PCR; amplification of HBV DNA and VHD RNA by conventional PCR; direct sequencing of both viruses; phylogenetic analysis and research of resistance mutations in rt HBV. As a result, a total of 30 reactive HBsAg and Total Anti-HD patients, all from the State of Amazonas, were included in the study. Of these, 9/30 had both plasma and hepatic tissue samples. RNA-VHD was detected in 13/30 (46.67%) and HBV DNA in 11/30 (36.67%). Of the 09 plasma and hepatic tissue samples, VHD-RNA was detected in 2/9 (22.27%) and HBV DNA in 8/9 (88.89%). Of the 30 plasma samples submitted to VHD RNA quantification, viral load was obtained in 13/30 (46.67%). We also analyzed the 9 samples of hepatic tissue, obtaining viral load in 2/9 (22.27%). In the phylogenetic analysis, all the Delta sequences clustered in the clade VHD-3, already those of HBV, the genotypes A 15/19 (78%), D 2/19 (11%) and F 2/19 (11%). Concerning the search for antiviral resistance mutations in rt-HBV, 3/11 (27%) and 5/08 (62%) sequences isolated from plasma and hepatic tissue, respectively, showed resistance mutations. Conclusions: three HBV genotypes A, D and F were detected, independent of the source material; all extracted from delta were exclusively VHD-3; the viral load of the delta virus was higher in those extracted from hepatic tissue; a high percentage of mutations in the HBV polymerase region was detected, especially in isolated hepatic tissue sequences. Finally, the profile of the HBV and VHD genotypes found in the study reflects the same pattern found in the region by other authors. O v??rus da hepatite B (VHB) ?? respons??vel por um grave problema de sa??de p??blica mundial, sendo que o problema torna-se ainda maior quando ocorre coinfec????o ou superinfec????o com o v??rus D (VHD). Estima-se que das 240 milh??es de pessoas no mundo portadoras cr??nicas de infec????o pelo VHB, 15 milh??es s??o portadoras de infec????o pelo VHD. Algumas regi??es da Bacia Amaz??nica, particularmente o Estado do Amazonas s??o consideradas ??reas end??micas de infec????o por esses v??rus. O VHD ?? classificado em oito diferentes tipos (VHD-1-VHD-8) e o VHB em dez (A-J). Sendo assim, esta pesquisa tem como objetivo, caracterizar os gen??tipos do v??rus da hepatite B e D no plasma e tecido hep??tico e investigar poss??vel associa????o dos gen??tipos com a evolu????o da doen??a. Foram analisadas amostras de plasma e de tecido hep??tico de pacientes portadores cr??nicos HBsAg reativos coinfectados com o VHD. Foi realizada a quantifica????o da carga viral do VHD no plasma por PCR em tempo real; amplifica????o do DNA-VHB e RNA-VHD por PCR convencional; sequenciamento direto de ambos os v??rus; an??lise filogen??tica e pesquisa de muta????es de resist??ncia na rt do VHB. Como resultados, foram inclu??dos no estudo um total de 30 pacientes HBsAg e Anti-HD Total reativos, todos procedentes do Estado do Amazonas. Destes, 9/30 tinham simultaneamente amostras de plasma e tecido hep??tico. O RNA-VHD foi detectado em 13/30 (46,67%) e o DNA-VHB em 11/30 (36,67%). Das 09 amostras de plasma e tecido hep??tico o RNA-VHD foi detectado em 02/9 (22,27%) e o DNA-VHB em 08/9 (88,89%). Das 30 amostras de plasma submetidas a quantifica????o do RNA VHD, obteve-se a carga viral de 13/30 (46,67%). Tamb??m analisamos as 9 mostras de tecido hep??tico, obtendo-se a carga viral em 2/9 (22,27%). Na an??lise filogen??tica, todas as sequ??ncias de Delta agruparam-se no clado VHD3, j?? as de VHB, foram identificados os gen??tipos A 15/19 (78%), D 2/19 (11%) e F 2/19 (11%). Em rela????o ?? pesquisa de muta????es de resist??ncia ?? antivirais na rt-VHB, verificou-se que 3/11 (27%) e 05/08 (62%) sequencias isoladas do plasma e tecido hep??tico, respectivamente, apresentavam muta????es de resist??ncia. Conclus??es: foi detectado tr??s gen??tipos do VHB A, D e F, independente do material de origem; todos os extra??dos de delta foram exclusivamente VHD-3; a carga viral do v??rus delta, foi maior nos extra??dos de tecido hep??tico; quanto ao perfil de muta????es de resist??ncia, foi detectado um percentual elevado de muta????es na regi??o da polimerase do VHB, principalmente nas sequ??ncias isoladas do tecido hep??tico. Por fim, o perfil dos gen??tipos do VHB e VHD encontrados no estudo reflete o mesmo padr??o encontrado na regi??o por outros autores