Julio Rendón

Virus–host interplay in hepatitis B virus infection and epigenetic treatment strategies

Worldwide, chronic hepatitis B virus (HBV) infection is a major health problem and no cure exists. Importantly, hepatocyte intrusion by HBV particles results in a complex deregulation of both viral and host cellular genetic and epigenetic processes. Among the attempts to develop novel therapeutic approaches against HBV infection, several options targeting the epigenomic regulation of HBV replication are gaining attention. These include the experimental treatment with ‘epidrugs’. Moreover, as a targeted approach, the principle of ‘epigenetic editing’ recently is being exploited to control viral replication. Silencing of HBV by specific rewriting of epigenetic marks might diminish viral replication, viremia, and infectivity, eventually controlling the disease and its complications. Additionally, epigenetic editing can be used as an experimental tool to increase our limited understanding regarding the role of epigenetic modifications in viral infections. Aiming for permanent epigenetic reprogramming of the viral genome without unspecific side effects, this breakthrough may pave the roads for an ambitious technological pursuit: to start designing a curative approach utilizing manipulative molecular therapies for viral infections in vivo.

Hepatitis E Virus Genotype 3 in Colombia: Survey in Patients with Clinical Diagnosis of Viral Hepatitis.

Background Hepatitis E virus is a major cause of outbreaks as well as sporadic hepatitis cases worldwide. The epidemiology of this enterically transmitted infection differs between developing and developed countries. The aims of this study were to describe HEV infection in Colombian patients and to characterize the genotype. Methods A prospective study was carried out on 40 patients aged over 15 with a clinical diagnosis of viral hepatitis, recruited from five primary health units in the city of Medellin, Colombia. Fecal samples obtained from the 40 consecutives cases were analyzed for HEV RNA using nested reverse transcription PCR for both ORF1 and ORF2-3. The amplicons were sequenced for phylogenetic analyses. Results Nine (22.5%) cases of HEV infection were identified in the study population. Three HEV strains obtained from patients were classified as genotype 3. No significant association was found between cases of Hepatitis E and the variables water drinking source, garbage collection system and contact with pigs. Conclusions This is the first prospective study of hepatitis E in Colombian patients. The circulation of the genotype 3 in this population is predictable considering the reports of the region and the identification of this genotype from pigs in the state of Antioquia, of which Medellin is the capital. Further studies are necessary to establish whether zoonotic transmission of HEV is important in Colombia.

Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia

Background Hepatitis B virus (HBV) occult infection (OBI) is a risk factor to be taken into account in transfusion, hemodialysis and organ transplantation. The aim of this study was to identify and characterize at the molecular level OBI cases in patients with end-stage liver disease. Methods Sixty-six liver samples were obtained from patients with diagnosis of end-stage liver disease submitted to liver transplantation in Medellin (North West, Colombia). Samples obtained from patients who were negative for the surface antigen of HBV (n = 50) were tested for viral DNA detection by nested PCR for ORFs S, C, and X and confirmed by Southern-Blot. OBI cases were analyzed by sequencing the viral genome to determine the genotype and mutations; additionally, viral genome integration events were examined by the Alu-PCR technique. Results In five cases out of 50 patients (10%) the criteria for OBI was confirmed. HBV genotype F (subgenotypes F1 and F3), genotype A and genotype D were characterized in liver samples. Three integration events in chromosomes 5q14.1, 16p13 and 20q12 affecting Receptor-type tyrosine-protein phosphatase T, Ras Protein Specific Guanine Nucleotide Releasing Factor 2, and the zinc finger 263 genes were identified in two OBI cases. Sequence analysis of the viral genome of the 5 OBI cases showed several punctual missense and nonsense mutations affecting ORFs S, P, Core and X. Conclusions This is the first characterization of OBI in patients with end-stage liver disease in Colombia. The OBI cases were identified in patients with HCV infection or cryptogenic cirrhosis. The integration events (5q14.1, 16p13 and 20q12) described in this study have not been previously reported. Further studies are required to validate the role of mutations and integration events in OBI pathogenesis.

Infección oculta por el virus de la hepatitis B en pacientes sometidos a trasplante hepático

Introduccion: la infeccion oculta por el virus de la hepatitis B (VHB) se caracteriza por la presencia del genoma viral en suero y/o tejido hepatico de individuos negativos para el antigeno de superficie HBsAg. La infeccion oculta se ha asociado con el desarrollo de cirrosis y carcinoma hepatocelular. Objetivo: identificar casos de infeccion oculta por el VHB en pacientes con diagnostico de cirrosis y carcinoma hepatocelular, sometidos a trasplante hepatico. Materiales y metodos: entre febrero de 2013 y marzo de 2014 fueron obtenidas muestras de explante hepatico provenientes de pacientes con diagnostico de cirrosis y/o carcino- ma hepatocelular. Se detecto el genoma del VHB mediante amplificacion de tres regiones del genoma viral (S, Core y X). Las muestras positivas se confirmaron por reaccion en cadena de la polimerasa (PCR) en tiempo real para la region S. Resultados: se analizaron 15 muestras de tejido hepatico, y en dos (13,3%) se detecto el genoma del VHB mediante PCR anidada para la region S y por PCR semianidada para la region X, resultado confirmado por PCR en tiempo real. Estas muestras provenian de pacientes negativos para los marcadores serologicos de infeccion por el VHB, anti-HBc y anti-HBs. Conclusion: la frecuencia de infeccion oculta reportada en este estudio es similar a lo reportado en Brasil en muestras de biopsias obtenidas de pacientes con hepatitis cronica. Estudios adicionales son necesarios para estimar la frecuencia de infeccion oculta por VHB (OBI) en pacientes con hepatopatias terminales en Colombia.

Re-expressing Epigenetically Silenced Genes by Inducing DNA Demethylation Through Targeting of Ten-Eleven Translocation 2 to Any Given Genomic Locus

Epigenetic editing is a novel methodology to modify the epigenetic landscape of any genomic location. As such, the approach might reprogram expression profiles, without altering the DNA sequence. Epigenetic alterations, including promoter hypermethylation, are associated with an increasing number of human diseases. To exploit this situation, epigenetic editing rises as a new alternative to specifically demethylate abnormally hypermethylated regions. Here, we describe a methodology to actively demethylate the hypermethylated ICAM-1 promoter. Reducing DNA methylation in our target region increased the expression of the ICAM-1 gene. As the ICAM-1 gene in our cell lines was highly methylated (up to 80%), this approach proves a robust manner to reduce methylation for hypermethylated regions. Epigenetic editing therefore not only provides an approach to address mechanisms of gene expression regulation, but also adds to the therapeutic toolbox as current inhibitors of epigenetic enzymes are limited by genome-wide effects.

Analysis of hepatitis B virus genotypes by restriction fragment length polymorphism

INTRODUCTION Ten viral genotypes (A-J) distributed in all continents have been described for hepatitis B virus (HBV). One of the methodologies for determining the viral genotype is the restriction fragment length polymorphism (RFLP) technique, a simple and relatively inexpensive method, albeit with some limitations. OBJECTIVE The initial objective of the project was to identify the HBV genotypes by RFLP in serum samples obtained from patients and blood donors. However, due to the discrepancies of RFLP patterns it was also necessary to perform phylogenetic genotyping and in silico analysis of HBV sequences. MATERIALS AND METHODS We obtained 56 serum samples. DNA extraction was followed by PCR amplification of a fragment of HBV ORF S. We analyzed PCR products by RFLP with AlwI, BsrI, CfrI, HpaII and StyI, and we sequenced some. We compared the patterns obtained with those in previous reports. We also performed RFLP analysis in silico since we found differences between the patterns expected and those obtainedResults: We identified genotypes A and F, subgenotype F3, in the samples. This result is in agreement with those of previous studies carried out in Colombia; indeed, subgenotype F3 is the most frequent in the Andean region of the country, while genotype A is the most frequent HBV genotype in the western region (department of Chocó). Based on the in silico analysis of 229 HBV sequences from GenBank and 11 sequences of this study, we identified the RLFP pattern for genotype F, subgenotype F3, and we described some modifications of genotype A RFLP patterns. CONCLUSIONS We identified the single nucleotide polymorphism pattern for genotype F, subgenotype F3, by in silico analysis and sequencing. Further robust in silico analyses are necessary to validate the RFLP patterns of HBV genotype and subgenotypes.