Prevention in Hepatology.

The prevention of liver disease has improved significantly in the last few decades, to the point that it can now be considered a true success story. The wide variety of interventions, including comprehensive vaccination strategies, novel medications, lifestyle changes, and even preventive surgeries, have reduced the morbidity and mortality of chronic liver diseases. However, the prevalence of chronic liver diseases is increasing worldwide. Currently, fatty liver disease alone is estimated to be present in as much as 30% of the adult population. Furthermore, there is a trend towards increasing incidences of chronic hepatitis B, and a global lack of success in efforts to eliminate chronic hepatitis C. Thus, improving and efficiently rolling out existing and successful prevention strategies for chronic liver diseases will play an essential role in healthcare throughout the upcoming decades. In this review, we summarize the current options and concepts for preventing chronic liver diseases, highlight their limitations, and provide an outlook on probable future developments to improve awareness, integrated care, and the analysis of big data.

Epidemiology and Genetic Diversity of Hepatitis B Virus and Hepatitis Delta Virus Infection in Indigenous Communities in Colombia.

Despite the universal vaccination program, there are still regions and territories with a high prevalence of Hepatitis B Virus infection (HBV), such as the Amazon basin, where several indigenous communities live. Additionally, Hepatitis Delta Virus (HDV) is a defective that requires the hepatitis B surface antigen (HBsAg) for the assembly and release of de novo viral particles. Therefore, hepatitis D could be the result of HBV/HDV coinfection or HDV superinfection in individuals with chronic hepatitis B. Among the high prevalence HDV populations are indigenous communities of America. This study aims to describe and characterize the frequency of HBV and HDV infection, viral genotypes and HBV immune escape mutants in indigenous populations from different regions of Colombia. The diagnosis of hepatitis B and hepatitis D was confirmed by serological markers. Moreover, the HBV and HDV genome were amplified by PCR and RT-PCR, respectively, and, subsequently, the phylogenetic analysis was performed. We characterized 47 cases of chronic hepatitis B, 1 case of reactivation and 2 cases of occult hepatitis B infection (OBI). Furthermore, a high prevalence of HDV infection was identified in the study population (29.33%, 22/75) and the circulation of several HBV genotypes and subgenotypes (F1b, F3, F4, and D). Interestingly, this is the first report of the HDV genotype I circulation in this country. These findings demonstrated that HBV and HDV infections are still public health problems in indigenous communities in Colombia.

Vertical Transmission of Hepatitis B Virus-An Update.

Hepatitis B virus (HBV) infection is a major public health problem in the world. Approximately 296 million people are chronically infected. In endemic areas, vertical transmission is a common route of transmission. There are several strategies for the prevention of HBV vertical transmission, such as antiviral treatment during the third trimester of pregnancy and immunoprophylaxis to newborns that includes the administration of hepatitis B immune globulin (HBIG) and an HBV vaccine. Despite this, immunoprophylaxis failure can occur in up to 30% of infants born to HBeAg-positive mothers and/or with high viral load. Therefore, management and prevention of HBV vertical transmission is of paramount significance. In this article, we provided a review of the epidemiology, mechanisms of pathogenesis and risk factors of vertical transmission, as well as the strategies implemented to prevent the infection.

Outbreak report of SARS-CoV-2 infection by airborne transmission: Epidemiologic and molecular evidence. / Reporte de un brote de infección por SARS-CoV-2 por transmisión aérea: evidencia epidemiológica y molecular

INTRODUCTION: It has been shown that the transmission of SARS-CoV-2 occurs mainly by air, and the risk of infection is greater in closed spaces. OBJECTIVE: To describe the epidemiology, virology and molecular characterization of a COVID-19 outbreak at a closed vaccination point during the third wave of SARS-CoV-2 in Colombia. MATERIALS AND METHODS: Diagnostic tests, interviews, sampling, cell cultures and viral sequencing were carried out, the latter being molecular characterization and lineage identification. RESULTS: Seven workers were positive for SARS-CoV-2; among these, 3 samples were analyzed, plus an additional sample belonging to the mother of the presumed index case; all samples were identified with lineage B.1.625, with a maximum of 2 nucleotides difference between them. CONCLUSIONS: Variant B.1.625 was identified as the cause of the COVID-19 outbreak, and a co-worker was also identified as the index case. Unexpectedly, attending a vaccination day became a risk factor for acquiring the infection.

Introducción. Se ha demostrado que la transmisión de SARS-CoV-2 se produce principalmente por vía aérea y el riesgo de infección es mayor en espacios cerrados con alta concentración de personas; este último factor se presentó en algunos de los puestos de vacunación de la ciudad de Medellín. Objetivo. Describir la epidemiología, virología y caracterización molecular de un brote de COVID-19 en un punto de vacunación cerrado durante la tercera ola de SARS-CoV-2 en Colombia. Materiales y métodos. Se realizaron test diagnósticos, entrevistas, toma de muestras, aislamiento viral y secuenciación genómica. Con esta última, se hizo la caracterización molecular y se identificó el linaje. Resultados. Siete trabajadores fueron positivos para SARS-CoV-2, y de estos, tres muestras fueron secuenciadas, más una muestra adicional perteneciente a la madre del presunto caso índice. Todas las muestras fueron identificadas con el linaje B.1.625, con un máximo de dos nucleótidos de diferencia entre ellas. Conclusiones. Se identificó la variante B.1.625 como la causante del brote de COVID-19, y también un compañero de trabajo fue identificado como el caso índice. De forma imprevista, asistir a una jornada de vacunación se convirtió en un factor de riesgo para adquirir la infección.

Novel Putative Tymoviridae-like Virus Isolated from Culex Mosquitoes in Colombia.

The family Tymoviridae comprises positive-sense RNA viruses, which mainly infect plants. Recently, a few Tymoviridae-like viruses have been found in mosquitoes, which feed on vertebrate sources. We describe a novel Tymoviridae-like virus, putatively named, Guachaca virus (GUAV), isolated from Culex pipiens and Culex quinquefasciatus species of mosquitoes and collected in the rural area of Santa Marta, Colombia. After a cytopathic effect was observed in C6/36 cells, RNA was extracted and processed through the NetoVIR next-generation sequencing protocol, and data were analyzed through the VirMAP pipeline. Molecular and phenotypic characterization of the GUAV was achieved using a 5'/3' RACE, transmission electron microscopy, amplification in vertebrate cells, and phylogenetic analysis. A cytopathic effect was observed in C6/36 cells three days post-infection. The GUAV genome was successfully assembled, and its polyadenylated 3' end was corroborated. GUAV shared only 54.9% amino acid identity with its closest relative, Ek Balam virus, and was grouped with the latter and other unclassified insect-associated tymoviruses in a phylogenetic analysis. GUAV is a new member of a family previously described as comprising plant-infecting viruses, which seem to infect and replicate in mosquitoes. The sugar- and blood-feeding behavior of the Culex spp., implies a sustained contact with plants and vertebrates and justifies further studies to unravel the ecological scenario for transmission.

Outbreak report of SARS-CoV-2 infection by airborne transmission: Epidemiologic and molecular evidence / Reporte de un brote de infección por SARS-CoV-2 por transmisión aérea: evidencia epidemiológica y molecular

Introduction: It has been shown that the transmission of SARS-CoV-2 occurs mainly by air, and the risk of infection is greater in closed spaces. Objective: To describe the epidemiology, virology and molecular characterization of a COVID-19 outbreak at a closed vaccination point during the third wave of SARS-CoV-2 in Colombia. Materials and methods: Diagnostic tests, interviews, sampling, cell cultures and viral sequencing were carried out, the latter being molecular characterization and lineage identification. Results: Seven workers were positive for SARS-CoV-2; among these, 3 samples were analyzed, plus an additional sample belonging to the mother of the presumed index case; all samples were identified with lineage B.1.625, with a maximum of 2 nucleotides difference between them. Conclusions: Variant B.1.625 was identified as the cause of the COVID-19 outbreak, and a co-worker was also identified as the index case. Unexpectedly, attending a vaccination day became a risk factor for acquiring the infection.

Introducción. Se ha demostrado que la transmisión de SARS-CoV-2 se produce principalmente por vía aérea y el riesgo de infección es mayor en espacios cerrados con alta concentración de personas; este último factor se presentó en algunos de los puestos de vacunación de la ciudad de Medellín. Objetivo. Describir la epidemiología, virología y caracterización molecular de un brote de COVID-19 en un punto de vacunación cerrado durante la tercera ola de SARS-CoV-2 en Colombia. Materiales y métodos. Se realizaron test diagnósticos, entrevistas, toma de muestras, aislamiento viral y secuenciación genómica. Con esta última, se hizo la caracterización molecular y se identificó el linaje. Resultados. Siete trabajadores fueron positivos para SARS-CoV-2, y de estos, tres muestras fueron secuenciadas, más una muestra adicional perteneciente a la madre del presunto caso índice. Todas las muestras fueron identificadas con el linaje B.1.625, con un máximo de dos nucleótidos de diferencia entre ellas. Conclusiones. Se identificó la variante B.1.625 como la causante del brote de COVID-19, y también un compañero de trabajo fue identificado como el caso índice. De forma imprevista, asistir a una jornada de vacunación se convirtió en un factor de riesgo para adquirir la infección.

Usefulness of an in vitro-transcribed RNA control for the detection and quantification of Yellow fever virus through real-time reverse transcription-polymerase chain reaction.

INTRODUCTION: Unvaccinated individuals in endemic areas with proven enzootic transmission of Yellow fever virus are at risk of infection due to a dramatic shift in the epidemiology of the disease over recent years. For this reason, epidemiological surveillance and laboratory confirmation of cases have become mandatory. OBJECTIVE: To develop and test a control RNA for YFV detection through real-time RT-PCR. METHODS: A 437-bp insert containing the T7 promoter and the target sequences for two different in-house protocols was designed in the context of the pUC57 vector and obtained through gene synthesis. After T7-driven in vitro transcription, standard curves were developed for Log10 serial dilutions of the YFV control RNA with 8 replicates. RESULTS: A dynamic range of quantification of 10 orders of magnitude was observed with a limit of detection of 6.3 GCE/µL (95% CI, 2.6 to 139.4 GCE/µL). CONCLUSION: The plasmid construct is available for YFV molecular test validation on clinical, entomological, and epizootic samples.

Reinfección por virus de la hepatitis C: revisión de tema y presentación de un caso / Hepatitis C virus reinfection: A review of the topic and case report

Resumen La infección crónica por el virus de la hepatitis C (VHC) afecta a 58 millones de personas y es una importante causa de morbimortalidad alrededor del mundo. La reinfección por VHC es un problema creciente en personas con factores de riesgo como consumo pesado de alcohol, sexo anal, sexo grupal y compartir agujas y jeringas; este tipo de infección se define como un nuevo contagio de VHC con un genotipo viral diferente al de la primera infección en un paciente luego de lograr una respuesta viral sostenida (RVS). La reinfección se presenta, en parte, debido a la ausencia de estrategias de promoción y prevención. Teniendo en cuenta estos antecedentes, se han propuesto estrategias más pragmáticas para controlar la infección por VHC y evitar la reinfección, tales como la microeliminación. En el presente artículo se presenta un caso de un paciente que presenta alteración en los marcadores de la bioquímica hepática, por lo que se solicita una prueba diagnóstica de infección por VHC y luego genotipificación viral, y se evidenció una infección por VHC genotipo 1, subgenotipo 1A. Se inició el manejo con antivirales de acción directa y se documentó una adecuada RVS12. Tres meses después el paciente regresó a consulta y en los exámenes de control se evidenció una carga viral elevada de VHC, por lo que se solicitó genotipificación y se demostró una nueva infección por VHC genotipo 4.

Abstract Chronic hepatitis C (HCV) infection affects 58 million people and is a significant cause of morbidity and mortality worldwide. HCV reinfection is a growing problem in people with risk factors such as heavy alcohol use, anal sex, group sex, and sharing needles and syringes. This type of infection is defined as a new HCV infection with a different viral genotype than the first infection in a patient after achieving a sustained viral response (SVR). Reinfection occurs, in part, due to the absence of promotion and prevention strategies. Taking this background into account, more pragmatic approaches have been proposed to control HCV infection and avoid reinfection, such as micro elimination. This article reports the case of a patient with alterations in biochemical liver markers, for which a diagnostic test for HCV infection and then viral genotyping was requested. Infection by HCV genotype 1, subgenotype 1A, was evidenced. Management with direct-acting antivirals was started, and an adequate SVR12 was documented. Three months later, the patient returned, and the control tests showed a high HCV viral load, for which genotyping was requested, showing a new HCV genotype 4 infection.

Detection of hepatitis E virus genotype 3 in wastewater by an electrochemical genosensor.

Hepatitis E Virus (HEV) is an etiologic agent of hepatitis worldwide. HEV genotype 3 is the most prevalent in non-endemic regions, identified in humans, pigs and environmental samples. Thus, considering the zoonotic nature of HEV genotype 3, viral genome detection in wastewater concerns public health authorities. Electrochemical biosensors are promising analytical tools for viral genome detection in outside settings. This work reports on a highly specific, sensitive and portable electrochemical genosensor to detect HEV genotype 3 in wastewater samples. Based on the alignment analysis of HEV genotype 3 genome sequences available in GenBank, highly specific DNA target probes were designed to hybridize a target sequence within the ORF2/ORF3 overlapping genome region of HEV in between a biotinylated capture probe and a signal probe labeled with digoxigenin, in a sandwich-type format. An anti-Dig antibody labeled with the horseradish peroxidase (HRP) enzyme allowed electrochemical detection. The specificity of the target molecular probes of the viral genome was determined before the biosensor assembly by in silico analysis, PCR and qPCR assays demonstrating efficient amplification of two targets, i.e., nucleotides 5338-5373 and 5328-5373, but this last one of higher performance. The electrochemical response of the genosensor with synthetic HEV was target concentration-dependent in a linear range from 300 pM to 2.4 nM, with a sensitivity of 16.93 µA/nM, a LOD 1.2 pM and high reproducibility. The genosensor response was differential when interrogated with the HEV genotype 3 viral genomes from wastewater against other four viruses. Therefore, the approach offers a step forward to the epidemiologic surveillance of viruses in wastewater as an early warning system.

Genetic diversity of hepatitis C virus and resistance associated substitutions to direct-acting antiviral treatment in Colombia.

Hepatitis C virus (HCV) infection is one of the leading risk factors for end-stage liver disease development worldwide. This RNA virus displays high genetic diversity with 8 genotypes and 96 subgenotypes with heterogeneous geographical distribution around the world. In this study, we carried out an active case finding of individuals with a history of transfusion events before 1996 in three cities in Colombia. Then, the characterization of the HCV genotypes, subgenotypes, and resistance associate substitutions (RAS) was performed in samples positives for antibodies anti-HCV + from this study population. In addition, samples from PWID and patients with end-stage liver disease submitted to liver transplantation were included in the phylogenetic and RAS analysis. The 5'UTR, NS5A, and NS5B regions of the HCV genome were amplified in serum or liver explants samples. After the edition, assembly, and alignment of the sequences, genotyping through phylogenetic analysis was performed using IQTREE V2.0.5 based on the maximum likelihood approach. The identification of RAS was carried out by alignments based on the reference sequence (GenBank NC_004102). Two hundred sixty individuals with blood transfusion events before 1996 were recruited. The seroprevalence of antibodies anti-HCV was 2.69% in this population. The HCV genotypes 1, 2, and 4 and subgenotypes 1a, 1b, 2a, 4a and 4d were characterized in samples of the study populations. Three RAS (Q30R, C316N, and Y93H) were identified in samples obtained from 2 individuals who received blood transfusion before 1996 and without previous antiviral treatment and 6 samples obtained from patients with end-stage liver disease. Among the 20 samples analyzed, the HCV genotype 1, subgenotype 1b, was the most frequent (60%). We report the first characterization of HCV subgenotypes 4a and 4d and the first RAS identification in patients in Colombia.

SARS-CoV-2 electrochemical immunosensor based on the spike-ACE2 complex.

Rapid, straightforward, and massive diagnosis of coronavirus disease 2019 (COVID-19) is one of the more important measures to mitigate the current pandemics. This work reports on an immunosensor to rapidly detect the spike protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The immunosensing device entraps the spike protein linked to angiotensin-converting enzyme host receptor (ACE2) protein in a sandwich between carboxylated magnetic beads functionalized with an anti-spike antibody and an anti-ACE2 antibody, further labeled with streptavidin (poly)horseradish peroxidase (HRP) reporter enzyme. The particles were confined at the surface of screen-printed gold electrodes, whose signal resulting from the interaction of the enzyme with a mediator was recorded in a portable potentiostat. The immunosensor showed a sensitivity of 0.83 µA∗mL/µg and a limit of detection of 22.5 ng/mL of spike protein, with high reproducibility. As a proof-of-concept, it detected commercial spike protein-supplemented buffer solutions, pseudovirions, isolated viral particles and ten nasopharyngeal swab samples from infected patients compared to samples from three healthy individuals paving the way to detect the virus closer to the patient.

[Epidemiology of hepatitis C virus infection in ColombiaEpidemiologia da infecção pelo vírus da hepatite C na Colômbia]. / Epidemiología de la infección por el virus de la hepatitis C en Colombia.

OBJECTIVE: To describe the epidemiology of hepatitis C virus (HCV) infection in Colombia. METHODS: Critical review of epidemiological studies of HCV infection in Colombia. The PubMed, SciELO, and ScienceDirect databases were searched for original articles and reviews on the subject published from 1989 to 2020. Reports from the National Institute of Health and the High Cost Account of the Ministry of Health and Social Protection were also reviewed. RESULTS: Data on seroprevalence of HCV antibodies in blood donors range from 1.5% to 0.32%, corresponding to reports at the beginning and end of the study period, respectively. In the population with risk factors, a high prevalence of HCV infection is observed, although with variations over time. With respect to HCV genotypes in Colombia, genotypes 1, 2, 3, and 4 (subtypes 1a, 1b, 2a, and 3a) have been identified. CONCLUSIONS: In the observation period, a decrease was seen in seroprevalence of HCV infection in blood donors and hemodialysis patients in Colombia, demonstrating the impact of safe blood policies and biosafety measures. Studies in people who inject illicit drugs indicate a high prevalence of infection, with regional differences within the country. HCV genotype 1, subtype 1b, is the most frequent in the different studies carried out in Colombia, and the most recent report of the High Cost Account of the Ministry of Health and Social Protection indicates that genotype 4 is the second most frequent genotype in the country.

OBJETIVO: Descrever a epidemiologia da infecção pelo vírus da hepatite C na Colômbia. MÉTODOS: Foi realizada uma revisão crítica de estudos epidemiológicos sobre a infecção pelo HCV na Colômbia por meio de busca de artigos originais e revisões a respeito do tema, publicados no período de 1989 a 2020, nos bancos de dados PubMed, SciELO e ScienceDirect. Também foram analisados os relatórios do Instituto Nacional de Saúde e da Conta de Alto Custo do Ministério da Saúde e Proteção Social. RESULTADOS: O índice de soroprevalência de anticorpos anti-HCV em doadores de sangue varia de 1,5% a 0,32%, correspondendo aos relatos do início e do final do período de estudo, respectivamente. Na população com fatores de risco, observa-se prevalência elevada de infecção pelo HCV, embora com variações ao longo do tempo. Em relação aos genótipos do HCV, foram identificados os genótipos 1, 2, 3 e 4 (subgenótipos 1a, 1b, 2a e 3a) na Colômbia. CONCLUSÕES: No período de interesse, observa-se uma diminuição da soroprevalência da infecção pelo HCV em doadores de sangue e em pacientes em tratamento hemodialítico na Colômbia, o que demonstra o impacto das políticas de sangue seguro e medidas de biossegurança. Estudos com usuários de drogas ilícitas injetáveis indicam alta prevalência de infecção, com diferenças de acordo com a região do país. O subgenótipo 1b do genótipo 1 do HCV é o mais prevalente nos diferentes estudos realizados na Colômbia, enquanto o informe mais recente da Conta de Alto Custo do Ministério da Saúde e Proteção Social indica que o genótipo 4 é o segundo mais frequente no país.

Epidemiología de la infección por el virus de la hepatitis C en Colombia

[RESUMEN]. Objetivo. Describir la epidemiología de la infección por el virus de la hepatitis C (VHC) en Colombia. Métodos. Revisión crítica de los estudios de epidemiología de la infección por VHC en Colombia mediante búsqueda de artículos originales y revisiones de tema publicados en el período 1989 a 2020 en las bases de datos PubMed, SciELO y ScienceDirect. Además, se revisaron los informes del Instituto Nacional de Salud y de la Cuenta de Alto Costo del Ministerio de Salud y Protección Social. Resultados. Los datos de seroprevalencia de anticuerpos anti-VHC en donantes de sangre están en un rango de 1,5% a 0,32%, que corresponden a los informes del inicio y el final del período de estudio, respectiva-mente. En la población con factores de riesgo se observa una alta prevalencia de infección por VHC, aunque con variaciones a lo largo del tiempo. Con respecto a los genotipos de VHC en Colombia, se han identificado los genotipos 1, 2, 3 y 4 (subgenotipos 1a, 1b, 2a y 3a). Conclusiones. En el período de observación, se describe una disminución en la seroprevalencia de la infección por VHC en donantes de sangre y en pacientes en tratamiento con hemodiálisis en Colombia, lo que demuestra el impacto de las políticas de sangre segura y las medidas de bioseguridad. Los estudios en personas que usan drogas ilícitas por vía inyectable indican una alta prevalencia de infección, con diferencias según la región del país. El genotipo 1, subgenotipo 1b, del VHC es el más frecuente en los distintos estudios realizados en Colombia, y el informe más reciente de la Cuenta de Alto Costo del Ministerio de Salud y Protección Social señala que el genotipo 4 es el segundo genotipo más frecuente en el país.

[ABSTRACT]. Objective. To describe the epidemiology of hepatitis C virus (HCV) infection in Colombia. Methods. Critical review of epidemiological studies of HCV infection in Colombia. The PubMed, SciELO, and ScienceDirect databases were searched for original articles and reviews on the subject published from 1989 to 2020. Reports from the National Institute of Health and the High Cost Account of the Ministry of Health and Social Protection were also reviewed. Results. Data on seroprevalence of HCV antibodies in blood donors range from 1.5% to 0.32%, correspon-ding to reports at the beginning and end of the study period, respectively. In the population with risk factors, a high prevalence of HCV infection is observed, although with variations over time. With respect to HCV genotypes in Colombia, genotypes 1, 2, 3, and 4 (subtypes 1a, 1b, 2a, and 3a) have been identified. Conclusions. In the observation period, a decrease was seen in seroprevalence of HCV infection in blood donors and hemodialysis patients in Colombia, demonstrating the impact of safe blood policies and biosafety measures. Studies in people who inject illicit drugs indicate a high prevalence of infection, with regional differences within the country. HCV genotype 1, subtype 1b, is the most frequent in the different studies carried out in Colombia, and the most recent report of the High Cost Account of the Ministry of Health and Social Protec-tion indicates that genotype 4 is the second most frequent genotype in the country.

[RESUMO]. Objetivo. Descrever a epidemiologia da infecção pelo vírus da hepatite C na Colômbia. Métodos. Foi realizada uma revisão crítica de estudos epidemiológicos sobre a infecção pelo HCV na Colômbia por meio de busca de artigos originais e revisões a respeito do tema, publicados no período de 1989 a 2020, nos bancos de dados PubMed, SciELO e ScienceDirect. Também foram analisados os relatórios do Instituto Nacional de Saúde e da Conta de Alto Custo do Ministério da Saúde e Proteção Social. Resultados. O índice de soroprevalência de anticorpos anti-HCV em doadores de sangue varia de 1,5% a 0,32%, correspondendo aos relatos do início e do final do período de estudo, respectivamente. Na população com fatores de risco, observa-se prevalência elevada de infecção pelo HCV, embora com variações ao longo do tempo. Em relação aos genótipos do HCV, foram identificados os genótipos 1, 2, 3 e 4 (subgenótipos 1a, 1b, 2a e 3a) na Colômbia. Conclusões. No período de interesse, observa-se uma diminuição da soroprevalência da infecção pelo HCV em doadores de sangue e em pacientes em tratamento hemodialítico na Colômbia, o que demonstra o impacto das políticas de sangue seguro e medidas de biossegurança. Estudos com usuários de drogas ilícitas injetáveis indicam alta prevalência de infecção, com diferenças de acordo com a região do país. O subgenótipo 1b do genótipo 1 do HCV é o mais prevalente nos diferentes estudos realizados na Colômbia, enquanto o informe mais recente da Conta de Alto Custo do Ministério da Saúde e Proteção Social indica que o genótipo 4 é o segundo mais frequente no país.

EXPRESIÓN DE LA PROTEÍNA CORE DEL VIRUS DE LA HEPATITIS C EN CÉLULAS HepG2 USANDO EL VIRUS DEL BOSQUE DE SEMLIKI / Hepatitis C virus core protein expression in HepG2 cells using Semliki Forest Virus

RESUMEN El Virus de la Hepatitis C (VHC) codifica la proteína Core. Que, además de ser la subunidad de la cápside, participa en diferentes mecanismos de patogénesis de la infección por VHC. Dado que el sistema de replicación in vitro del VHC presenta limitaciones, el uso de vectores virales podría ser una herramienta útil para estudiar las propiedades de la proteína Core. Con el fin de validar el vector con el Virus del Bosque de Semliki (SFV) para el estudio de Core en células HepG2, se evaluó la expresión de la proteína verde fluorescente (GFP) y la proteína Core utilizando este vector viral. Las expresiones de GFP y Core se detectaron en células HepG2 transducidas con rSFV de 24 a 96 horas postransducción. La expresión de la proteína Core fue inferior a la expresión de GFP en las células HepG2. Teniendo en cuenta que la proteína Core del VHC puede regular la actividad del gen p53, se evaluó el nivel transcripcional de este gen. Se observó una disminución en el nivel de mARN de p53 en las células luego de la transducción, comparado con las células control. Aunque las células transducidas con rSFV-Core presentaron el menor nivel de mARN de p53, la diferencia no fue significativa comparada con las células transducidas con rSFV-GFP. Los resultados confirman que rSFV permite la expresión transitoria de proteínas heterólogas en líneas celulares de hepatoma humano. Se necesitan estudios adicionales para determinar si la expresión disminuida de Core puede deberse a degradación de la proteína viral.

ABSTRACT The Hepatitis C Virus (HCV) encodes the structural protein Core, which in addition to being the capsid subunit, participates in different mechanisms of HCV infection pathogenesis. Since HCV in vitro replication system has limitations, the use of viral vectors could be a useful tool to study the Core protein properties. To validate the Semliki Forest Virus (SFV) strategy in transduced HepG2 cells to study the HCV Core protein, the expression of green fluorescent protein (GFP) and Core protein expressions were detected 24 to 96 hours post-transduction in HepG2 cells transduced with rSFV. Core protein expression was lower than GFP expression in HepG2 cells. Since HCV Core protein can regulate the activity of the p53 gene, the transcriptional level of this gene was evaluated. A decrease in the level of p53 mRNA was observed in the cells after transduction, compared to the control cells. Although the cells transduced with rSFV-Core had the lowest level of p53 mRNA, the difference was not significant compared to cells transduced with rSFV-GFP. The results confirm that rSFV allows the transient expression of heterologous proteins in human hepatoma cell lines. Additional studies are needed to determine whether the decreased expression of Core may be due to the degradation of the viral protein.

Hepatitis C Virus Proteins Core and NS5A Are Highly Sensitive to Oxidative Stress-Induced Degradation after eIF2α/ATF4 Pathway Activation.

Hepatitis C virus (HCV) infection is accompanied by increased oxidative stress and endoplasmic reticulum stress as a consequence of viral replication, production of viral proteins, and pro-inflammatory signals. To overcome the cellular stress, hepatocytes have developed several adaptive mechanisms like anti-oxidant response, activation of Unfolded Protein Response and autophagy to achieve cell survival. These adaptive mechanisms could both improve or inhibit viral replication, however, little is known in this regard. In this study, we investigate the mechanisms by which hepatocyte-like (Huh7) cells adapt to cellular stress in the context of HCV protein overexpression and oxidative stress. Huh7 cells stably expressing individual HCV (Core, NS3/4A and NS5A) proteins were treated with the superoxide anion donor menadione to induce oxidative stress. Production of reactive oxygen species and activation of caspase 3 were quantified. The activation of the eIF2α/ATF4 pathway and changes in the steady state levels of the autophagy-related proteins LC3 and p62 were determined either by quantitative polymerase chain reaction (qPCR) or Western blotting. Huh7 cells expressing Core or NS5A demonstrated reduced oxidative stress and apoptosis. In addition, phosphorylation of eIF2α and increased ATF4 and CHOP expression was observed with subsequent HCV Core and NS5A protein degradation. In line with these results, in liver biopsies from patients with hepatitis C, the expression of ATF4 and CHOP was confirmed. HCV Core and NS5A protein degradation was reversed by antioxidant treatment or silencing of the autophagy adaptor protein p62. We demonstrated that hepatocyte-like cells expressing HCV proteins and additionally exposed to oxidative stress adapt to cellular stress through eIF2a/ATF4 activation and selective degradation of HCV pro-oxidant proteins Core and NS5A. This selective degradation is dependent on p62 and results in increased resistance to apoptotic cell death induced by oxidative stress. This mechanism may provide a new key for the study of HCV pathology and lead to novel clinically applicable therapeutic interventions.

Infección por el virus de la hepatitis delta / Hepatitis delta virus infection

El virus de la hepatitis delta (VHD) es un virus satélite del virus de la hepatitis B (VHB), dado que requiere el antígeno de superficie del VHB (HBsAg) para la producción de partículas virales infecciosas. Se han caracterizado ocho genotipos del VHD, con una distribución geográfica relacionada con la prevalencia de la infección por VHB. Se estima que aproximadamente el 5% de los pacientes con infección crónica por VHB también están infectados con VHD. Se han descrito dos tipos de infección: la coinfección simultánea por VHB y VHD, y la superinfección con VHD en un paciente previamente infectado por VHB, esta última asociada a una mayor morbilidad y mortalidad por falla hepática aguda. La infección se diagnostica en nuestro medio con la determinación de IgM contra el VHD, acompañada idealmente de la carga viral. Aunque el tratamiento de elección es la terapia con interferón alfa pegilado, en el momento se están evaluando otros medicamentos antivirales en ensayos clínicos, con resultados alentadores, teniendo en cuenta el efecto observado en la carga viral del VHD y/o del VHB en los pacientes. La presente revisión tiene como objetivo incluir temas como la biología del virus, la epidemiología, las características clínicas, el diagnóstico y el tratamiento en la infección por VHD.

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV), as it requires the HBV surface antigen (HBsAg) for the production of infectious viral particles. Eight HDV genotypes have been characterized with a geographic distribution associated with the prevalence of HBV infection. It is estimated that approximately 5% of patients with chronic HBV infection are also infected with HDV. Two types of infection have been described: coinfection, by simultaneously contracting HBV and HDV infection, and superinfection, by contracting HDV when chronically infected with HBV, the latter associated with increased morbidity and mortality from acute liver failure. Anti-HDV IgM is used to diagnose the infection, ideally together with the detection of HDV-RNA. Although the treatment of choice is pegylated interferon alpha, other antiviral drugs are currently being evaluated in clinical trials, with encouraging results, in terms of their effect on HDV and/or HBV viral load in patients. This review aims to include topics such as virus biology, epidemiology, clinical manifestations, diagnosis, and treatment in HDV infection.

Ausencia de expresión de la proteína Core del virus de la hepatitis C en células dendríticas derivadas de monocitos humanos utilizando virus del Bosque de Semliki recombinante / Lack of expression of hepatitis c virus core protein in human monocyte-derived dendritic cells using recombinant semliki forest virus

ABSTRACT Hepatitis C Virus belongs to the Flaviviridae family. One proposed mechanism of HCV persistence in the ability to infect hematopoietic cells, including Dendritic cells (DCs). HCV infection of DCs could impair their functions that represent one of the mechanisms, thus hampering viral clearance by the host immune system. Among HCV-encoded proteins, the highly conserved Core protein has been suggested to be responsible for the immunomodulatory properties of this Hepacivirus. Recombinant viral vectors expressing the HCV Core protein and allowing its transduction and therefore the expression of the protein into DCs could be useful tools for the analysis of the properties of the Core protein. Vaccinia Virus and retrovirus have been used to transduce human DCs. Likewise, gene transfer into DCs using Semliki Forest Virus has been reported. This study aimed to express the HCV Core protein in human monocyte-derived DCs using an SFV vector, in which the subgenomic RNA encoding the structural proteins was replaced by the HCV Core sequence and then analyze the effects of its expression on DCs functions.

RESUMEN El virus de la Hepatitis C (VHC) pertenece a la familia Flaviviridae. Uno de los mecanismos propuestos de la persistencia del VHC es la capacidad de infectar células hematopoyéticas, incluidas las células dendríticas (DCs). La infección por VHC de DCs podría alterar sus funciones y corresponde a uno de los mecanismos que impiden el aclaramiento de la infección por VHC por el sistema inmunitario del hospedero. Entre las proteínas codificadas por el VHC, se ha sugerido que la proteína Core, altamente conservada, es responsable de las propiedades inmunomoduladoras de este Hepacivirus. Los vectores virales recombinantes que expresan la proteína Core y permiten su transducción a DCs podrían ser herramientas útiles para el análisis de las propiedades de esta proteína. El virus Vaccinia y el retrovirus se han utilizado para la transducción de DCs humanas. Del mismo modo, la transducción de DCs usando el virus del bosque de Semliki ha sido reportada. El objetivo de este estudio fue expresar la proteína Core de VHC en DCs derivadas de monocitos humanos utilizando un vector de SFV, en el que el ARN subgenómico que codifica las proteínas estructurales fue reemplazado por la secuencia Core del VHC y evaluar los efectos de su expresión en las funciones de DCs.

Mutations in the Helicobacter pylori 23S rRNA gene associated with clarithromycin resistance in patients at an endoscopy unit in Medellín, Colombia / Mutaciones del gen ARN ribosómico 23S de Helicobacter pylori asociadas con resistencia a claritromicina en pacientes atendidos en una unidad de endoscopia de Medellín, Colombia

Introduction: Clarithromycin is the first-line antibiotic for the treatment of Helicobacter pylori infection. Bacterial resistance is mainly due to the presence of specific mutations in the 23S ribosomal RNA (rRNA) gene. Objective: To determine the frequency of A2143G and A2142G specific mutations in the 23S rRNA gene associated with clarithromycin resistance of H. pylori in samples from patients with dyspeptic manifestations in Medellín, northwestern Colombia. Materials and methods: DNA was extracted from gastric biopsy samples of patients with dyspeptic manifestations seen at an endoscopy unit in Medellín between 2016 and 2017. PCR was performed to amplify the bacterial s and m vacA regions, and a region in the 23S rRNA gene. The presence of the A2142G and A2143G mutations was determined using the restriction fragment length polymorphism (RFLP) technique with the BbsI and BsaI enzymes, respectively. Results: The prevalence of infection was 44.2% (175/396), according to the histopathology report. The positive samples were analyzed and the three regions of the bacterial genome were amplified in 143 of the 175 samples. The A2143G and A2142G mutations were identified in 27 samples (18.8%, 27/143). The most frequent mutation was A2143G (81.5%, 22/27). Conclusions: We found a high prevalence of H. pylori mutations associated with clarithromycin resistance in the study population. Further studies are required to determine the bacterial resistance in the Colombian population in order to define first line and rescue treatments.

Introducción. La claritromicina es el antibiótico de primera línea para el tratamiento de la infección por Helicobacter pylori. La resistencia bacteriana se produce principalmente por mutaciones puntuales del gen ARN ribosómico 23S (ARNr 23S). Objetivo. Determinar la frecuencia de las mutaciones puntuales A2143G y A2142G del gen ARNr 23S asociadas con la resistencia de H. pylori a la claritromicina en muestras de pacientes con manifestaciones dispépticas en Medellín, región noroccidental de Colombia. Materiales y métodos. Se extrajo ADN a partir de muestras de biopsia gástrica obtenidas de pacientes con manifestaciones dispépticas atendidos en una unidad de endoscopia entre el 2016 y el 2017. Mediante reacción en cadena de la polimerasa (PCR), se amplificaron las regiones s y m del gen vacA y una región del gen ARNr 23S bacteriano. La presencia de las mutaciones A2142G y A2143G se determinó por la técnica de polimorfismos de longitud de fragmentos de restricción (RFLP) con las enzimas BbsI y BsaI, respectivamente. Resultados. Se encontró una prevalencia de infección de 44,2 % (175/396), según el informe de histopatología. En 143 de estas 175 muestras positivas se amplificaron las tres regiones del genoma bacteriano. Se identificaron las mutaciones A2143G y A2142G en 27 muestras (18,8 %; 27/143), la mutación más frecuente fue la A2143G (81,5 %; 22/27). Conclusiones. Hubo una gran prevalencia de mutaciones asociadas con la resistencia de H. pylori a la claritromicina en la población de estudio. Se requieren estudios adicionales para establecer la resistencia bacteriana en la población colombiana y, así, determinar los tratamientos de primera línea y de rescate.

Mutaciones del gen ARN ribosómico 23S de Helicobacter pylori asociadas con resistencia a claritromicina en pacientes atendidos en una unidad de endoscopia de Medellín, Colombia / Mutations in the Helicobacter pylori 23S rRNA gene associated with clarithromycin resistance in patients at an endoscopy unit in Medellín, Colombia

Resumen Introducción. La claritromicina es el antibiótico de primera línea para el tratamiento de la infección por Helicobacter pylori. La resistencia bacteriana se produce principalmente por mutaciones puntuales del gen ARN ribosómico 23S (ARNr 23S). Objetivo. Determinar la frecuencia de las mutaciones puntuales A2143G y A2142G del gen ARNr 23S asociadas con la resistencia de H. pylori a la claritromicina en muestras de pacientes con manifestaciones dispépticas en Medellín, región noroccidental de Colombia. Materiales y métodos. Se extrajo ADN a partir de muestras de biopsia gástrica obtenidas de pacientes con manifestaciones dispépticas atendidos en una unidad de endoscopia entre el 2016 y el 2017. Mediante reacción en cadena de la polimerasa (PCR), se amplificaron las regiones s y m del gen vacA y una región del gen ARNr 23S bacteriano. La presencia de las mutaciones A2142G y A2143G se determinó por la técnica de polimorfismos de longitud de fragmentos de restricción (RFLP) con las enzimas BbsI y BsaI, respectivamente. Resultados. Se encontró una prevalencia de infección de 44,2 % (175/396), según el informe de histopatología. En 143 de estas 175 muestras positivas se amplificaron las tres regiones del genoma bacteriano. Se identificaron las mutaciones A2143G y A2142G en 27 muestras (18,8 %; 27/143), la mutación más frecuente fue la A2143G (81,5 %; 22/27). Conclusiones. Hubo una gran prevalencia de mutaciones asociadas con la resistencia de H. pylori a la claritromicina en la población de estudio. Se requieren estudios adicionales para establecer la resistencia bacteriana en la población colombiana y, así, determinar los tratamientos de primera línea y de rescate.

Abstract Introduction: Clarithromycin is the first-line antibiotic for the treatment of Helicobacter pylori infection. Bacterial resistance is mainly due to the presence of specific mutations in the 23S ribosomal RNA (rRNA) gene. Objective: To determine the frequency of A2143G and A2142G specific mutations in the 23S rRNA gene associated with clarithromycin resistance of H. pylori in samples from patients with dyspeptic manifestations in Medellín, northwestern Colombia. Materials and methods: DNA was extracted from gastric biopsy samples of patients with dyspeptic manifestations seen at an endoscopy unit in Medellín between 2016 and 2017. PCR was performed to amplify the bacterial s and m vacA regions, and a region in the 23S rRNA gene. The presence of the A2142G and A2143G mutations was determined using the restriction fragment length polymorphism (RFLP) technique with the BbsI and BsaI enzymes, respectively. Results: The prevalence of infection was 44.2% (175/396), according to the histopathology report. The positive samples were analyzed and the three regions of the bacterial genome were amplified in 143 of the 175 samples. The A2143G and A2142G mutations were identified in 27 samples (18.8%, 27/143). The most frequent mutation was A2143G (81.5%, 22/27). Conclusions: We found a high prevalence of H. pylori mutations associated with clarithromycin resistance in the study population. Further studies are required to determine the bacterial resistance in the Colombian population in order to define first line and rescue treatments.