RESUMO
Hepatitis B virus (HBV) infects the liver resulting in end stage liver disease, cirrhosis, and hepatocellular carcinoma. Despite an effective vaccine, HBV poses a serious health problem globally, accounting for 257 million chronic carriers. Unique features of HBV, including its narrow virus-host range and its hepatocyte tropism, have led to major challenges in the development of suitable in vivo and in vitro model systems to recapitulate the HBV replication cycle and to test various antiviral strategies. Moreover, HBV is classified into at least nine genotypes and 35 sub-genotypes with distinct geographical distributions and prevalence, which have different natural histories of infection, clinical manifestation, and response to current antiviral agents. Here, we review various in vitro systems used to study the molecular biology of the different (sub)genotypes of HBV and their response to antiviral agents, and we discuss their strengths and limitations. Despite the advances made, no system is ideal for pan-genotypic HBV research or drug development and therefore further improvement is required. It is necessary to establish a centralized repository of HBV-related generated materials, which are readily accessible to HBV researchers, with international collaboration toward advancement and development of in vitro model systems for testing new HBV antivirals to ensure their pan-genotypic and/or customized activity.
Assuntos
Genótipo , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , DNA Viral , Suscetibilidade a Doenças , Genoma Viral , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Cultura Primária de Células , Fenômenos Fisiológicos ViraisRESUMO
Hepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established an in vitro replication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Replicon/efeitos dos fármacos , Replicon/genética , Substituição de Aminoácidos/efeitos dos fármacos , Substituição de Aminoácidos/genética , Linhagem Celular , Genótipo , Humanos , Mutação/efeitos dos fármacos , Mutação/genética , RNA Viral/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
The antibiotic resistance profiles of Escherichia coli (E. coli); isolated from different water sources in the Mmabatho locality were evaluated. Water samples were collected from the local wastewater- and water-treatment plants; the Modimola Dam and homes in the area; and then analysed for the presence of E. coli; using standard methods. Presumptive isolates obtained were confirmed by the analytical profile index test. Antibiotic susceptibility testing was performed by the disc diffusion method. Of the 230 E. coli isolates tested; marked antibiotic resistances (over 70) were observed for erythromycin; tetracycline; ampicillin; chloramphenicol and norfloxacin. Multiple antibiotic resistance patterns were also compiled. Overall; the phenotype T-Ap-E was frequent for E. coli isolated from the local wastewater and water-treatment plants; Modimola Dam and tap water. Cluster analysis performed showed a unique antibiotic resistance pattern which suggested a link between isolates from all sampling points. The findings indicated that improper wastewater treatment may have a potential impact on the dissemination and survival of E. coli; as well as other pathogenic bacteria in water for human and animal consumption. This may result in water- and food-borne disease outbreaks with a negative effect on antibiotic therapy
