Genetic variability in coding regions of the surface antigen and reverse transcriptase domain of hepatitis B virus polymerase, Colombia, 2002-2014

Introduction: Despite the availability of an effective vaccine and treatment to reduce the viral load and progressive hepatocellular injury, approximately 240 million people worldwide are chronically infected with the hepatitis B virus (HBV). In Colombia, the circulation of different viral genotypes has been confirmed. Mutations in the genome have been associated to antiviral therapy resistance, viral escape to neutralizing antibodies, occult infection and progression to hepatocellular carcinoma. Objective: To identify the genotypes and the presence of mutations in the coding region of the surface (S) antigen and the reverse transcriptase (RT) domain of the polymerase of HBV obtained from serum samples for hepatitis B diagnosis received by the Instituto Nacional de Salud during the period 2002-2014. Materials and methods: A total of 495 serum samples with previous HBsAg reactive result were used for molecular detection. A fragment of 1,591 nucleotides was sequenced, and the corresponding phylogenetic analysis was performed. Results: We detected the viral genome of HBV in 66 samples and 28 were successfully sequenced. The phylogenetic analysis allowed the identification of subgenotypes F3 and A2. The L180M and M204V resistance mutations were simultaneously identified in one sample, while the I169L resistance mutation was identified in another one. A single escape mutation, P120Q, was identified in one more. Two samples showed a deletion of 105 nucleotides in the preS1-preS2 region. Conclusions: The circulation of genotypes/subgenotypes F3 and A2 of HBV in Colombia was corroborated, as well as the presence of some resistance and escape mutations. The present study constitutes a contribution to the molecular epidemiology of HBV in Colombia

Recomendaciones para la detección y el diagnóstico por laboratorio de infecciones por arbovirus en la Región de las Américas

Los virus transmitidos por artrópodos (arbovirus) infectan a los seres humanos principalmente a través de la picadura de artrópodos hematófagos (por ejemplo, mosquitos, garrapatas y jejenes). Hay más de 100 arbovirus que causan infección y enfermedad en los seres humanos. Estas infecciones pueden ser desde asintomáticas hasta poner en riesgo la vida (1). Los arbovirus constituyen un grupo polifilético de virus de varias familias y géneros (1), e incluyen Flavivirus (familia Flaviviridae), Alphavirus (familia Togaviridae), Orthobunyavirus (familia Peribunyaviridae), Phlebovirus (familia Phenuiviridae) y Coltivirus (familia Reoviridae). La mayoría tiene un genoma de ARN monocatenario (de sentido positivo para las familias Flaviviridae y Togaviridae y negativo para la familia Peribunyaviridae y Phenuiviridae), mientras que los de la familia Reoviridae tienen ácido ribonucleico (ARN) bicatenario (1). Los arbovirus más importantes en las Américas son los flavivirus, entre ellos, los virus del dengue (DENV, por su sigla en inglés), del Zika (ZIKV, por su sigla en inglés), de la fiebre amarilla (YFV, por su sigla en inglés), del Nilo occidental (WNV, por su sigla en inglés) y de la encefalitis de San Luis (SLEV, por su sigla en inglés). También son frecuentes los alfavirus, entre los que se encuentran los virus del chikunguña (CHIKV, por su sigla en inglés), Mayaro (MAYV, por su sigla en inglés) y los de las encefalitis equinas, y el virus Oropouche (OROV, por su sigla en inglés), del género Orthobunyavirus. Esta publicación se elaboró como parte de la Estrategia para la prevención y el control de las enfermedades arbovirales, adoptada por el 55.o Consejo Directivo de la Organización Panamericana de la Salud (OPS) en septiembre del 2016 (2). En particular, se refiere a la cuarta línea estratégica de acción, que destaca la importancia de reforzar la capacidad técnica de la Red de Laboratorios de Diagnóstico de Arbovirus en la Región de las Américas (RELDA) (2). El propósito de esta publicación es brindar recomendaciones técnicas a los laboratorios de salud pública de las Américas sobre las muestras y pruebas que se usan en los laboratorios de vigilancia de las arbovirosis y la interpretación de sus resultados. El documento también le será útil a los centros de investigación y laboratorios de universidades que tengan que identificar infecciones arbovirales como parte de su investigación, para contribuir a la caracterización de los arbovirus y al entendimiento de su dinámica de transmisión.Agradecimientos. -- Siglas. -- Introducción. -- 1 Emergencia y ciclo de vida de los arbovirus. -- 1.1 Estructura, genoma y ciclo de vida de los arbovirus. -- 1.2 Origen y dinámica evolutiva de los arbovirus. -- 2 Toma, conservación y transporte de muestras. -- 2.1 Muestras para el diagnóstico de infecciones arbovirales . -- 2.2 Información epidemiológica y clínica adjunta a las muestras. -- 2.3 Envío de muestras al laboratorio. -- 2.4 Recepción y manipulación de las muestras en el laboratorio. -- 2.5 Envío de muestras a los laboratorios de referencia. -- 3 Bioseguridad y flujo de trabajo en el laboratorio. -- 3.1 Consideraciones sobre bioseguridad. -- 3.2 Flujo de trabajo en el laboratorio. -- 4 Diagnóstico por laboratorio del dengue, el chikunguña, el Zika y la fiebre amarilla. -- 4.1 Diagnóstico virológico: métodos directos. -- 4.2 Diagnóstico serológico: métodos indirectos. -- 4.3 Algoritmos recomendados para el diagnóstico por laboratorio de infecciones por los virus del dengue, el chikunguña, el Zika y la fiebre amarilla. -- 5 Diagnóstico por laboratorio de infecciones por los virus Mayaro y Oropouche. -- 5.1 Infección por el virus Mayaro . -- 5.2 Infección por el virus Oropouche. -- 6. Diagnóstico por laboratorio de otras infecciones arbovirales neurotrópicas. -- 6.1 Métodos virológicos. -- 6.2 Métodos serológicos. -- 7. Caracterización de genotipos y linajes por secuenciación nucleotídica. -- Referencias.https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=00003185070000-0002-8093-0544https://scienti.minciencias.gov.co/gruplac/jsp/visualiza/[email protected]://scholar.google.com.co/citations?user=cU2KyT4AAAAJ&hl=e

Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform

Artículo científico publicado en revista indexada en Scopus y categorizada en Q2.Dengue viruses (DENV) have become the most important arthropod-borne viruses, causing dengue and severe dengue fever in at least 50-100 million cases each year, mainly in tropical and subtropical countries. During recent years, important advances in the molecular biology concerning the life cycle of these viruses have allowed the manipulation and generation of recombinant viruses and replicons with multiple applications, mainly in viral biology and the screening of antiviral compounds. In the present study, we describe the construction of an enhanced green fluorescent protein-bearing DENV replicon under the control of the cytomegalovirus immediate early promoter. Following a rational in silico design and cloning by standard molecular biology techniques, a reporter DENV-2 replicon and a replication-deficient mutant were constructed, and characterized by confocal microscopy and real-time RT-PCR. The results showed successful transcription, translation, and autonomous viral RNA replication of the DENV replicon from its DNA clone. This novel DENV replicon will allow the study of viral replication and testing of antiviral candidates without the need for in vitro [email protected]

Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform

Dengue viruses (DENV) have become the most important arthropod-borne viruses, causing dengue and severe dengue fever in at least 50-100 million cases each year, mainly in tropical and subtropical countries. During recent years, important advances in the molecular biology concerning the life cycle of these viruses have allowed the manipulation and generation of recombinant viruses and replicons with multiple applications, mainly in viral biology and the screening of antiviral compounds. In the present study, we describe the construction of an enhanced green fluorescent protein-bearing DENV replicon under the control of the cytomegalovirus immediate early promoter. Following a rational in silico design and cloning by standard molecular biology techniques, a reporter DENV-2 replicon and a replication-deficient mutant were constructed, and characterized by confocal microscopy and real-time RT-PCR. The results showed successful transcription, translation, and autonomous viral RNA replication of the DENV replicon from its DNA clone. This novel DENV replicon will allow the study of viral replication and testing of antiviral candidates without the need for in vitro transcription. © 2017 S. Karger AG, [email protected]

Efficient method for molecular characterization of the 5' and 3' ends of the dengue virus genome

Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5' and 3' ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 5' and 3' ends of dengue virus types 1 to 4. The 5' and 3' ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 5' end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 3' end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 5' and 3' ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods. © 2020 by the authors. Licensee MDPI, Basel, [email protected]

Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome

Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 50 and 30 ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 50 and 30 ends of dengue virus types 1 to 4. The 50 and 30 ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 50 end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 30 end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 50 and 30 ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000318507https://orcid.org/0000-0002-8093-0544https://scienti.minciencias.gov.co/gruplac/jsp/visualiza/visualizagr.jsp?nro=00000000008981jose.usmec@campusucc.edu.cohttps://scholar.google.com.co/citations?user=cU2KyT4AAAAJ&hl=e

Molecular characterization of dengue virus reveals regional diversification of serotype 2 in Colombia

Dengue is hyperendemic in Colombia, where a cyclic behavior of serotype replacement leading to periodic epidemics has been observed for decades. This level of endemicity favors accumulation of dengue virus genetic diversity and could be linked to disease outcome. To assess the genetic diversity of dengue virus type 2 in Colombia, we sequenced the envelope gene of 24 virus isolates from acute cases of dengue or severe dengue fever during the period 2013-2016. The phylogenetic analysis revealed the circulation of the Asian-American genotype of dengue virus type 2 in Colombia during that period, the intra-genotype variability leading to divergence in two recently circulating lineages with differential geographic distribution, as well as the presence of nonsynonymous substitutions accompanying their emergence and diversification. © 2019 The Author(s)[email protected]

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.https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=00003185070000-0002-8093-0544https://scienti.minciencias.gov.co/gruplac/jsp/visualiza/visualizagr.jsp?nro=00000000008981jose.usmec@campusucc.edu.cohttps://scholar.google.com.co/citations?user=cU2KyT4AAAAJ&hl=e

Diversity and interactions among triatomine bugs, their blood feeding sources, gut microbiota and Trypanosoma cruzi in the Sierra Nevada de Santa Marta in Colombia

Chagas disease remains a major neglected disease in Colombia. We aimed to characterize Trypanosoma cruzi transmission networks in the Sierra Nevada de Santa Marta (SNSM) region, to shed light on disease ecology and help optimize control strategies. Triatomines were collected in rural communities and analyzed for blood feeding sources, parasite diversity and gut microbiota composition through a metagenomic and deep sequencing approach. Triatoma dimidiata predominated, followed by Rhodnius prolixus, Triatoma maculata, Rhodnius pallescens, Panstrongylus geniculatus and Eratyrus cuspidatus. Twenty-two species were identified as blood sources, resulting in an integrated transmission network with extensive connectivity among sylvatic and domestic host species. Only TcI parasites were detected, predominantly from TcIb but TcIa was also reported. The close relatedness of T. cruzi strains further supported the lack of separate transmission cycles according to habitats or triatomine species. Triatomine microbiota varied according to species, developmental stage and T. cruzi infection. Bacterial families correlated with the presence/absence of T. cruzi were identified. In conclusion, we identified a domestic transmission cycle encompassing multiple vector species and tightly connected with sylvatic hosts in the SNSM region, rather than an isolated domestic transmission cycle. Therefore, integrated interventions targeting all vector species and their contact with humans should be considered.https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0001344825https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000009091https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000318507https://orcid.org/0000-0003-0445-5814https://orcid.org/0000-0002-8093-0544https://scienti.minciencias.gov.co/gruplac/jsp/visualiza/visualizagr.jsp?nro=00000000008981andres.rojasgu@[email protected]@ucc.edu.cohttps://scholar.google.es/citations?hl=es&user=gwBokScAAAAJhttps://scholar.google.es/citations?hl=es&user=1pfSvNUAAAAJhttps://scholar.google.es/citations?hl=es&user=cU2KyT4AAAA

Diversity and interactions among triatomine bugs, their blood feeding sources, gut microbiota and Trypanosoma cruzi in the Sierra Nevada de Santa Marta in Colombia

Chagas disease remains a major neglected disease in Colombia. We aimed to characterize Trypanosoma cruzi transmission networks in the Sierra Nevada de Santa Marta (SNSM) region, to shed light on disease ecology and help optimize control strategies. Triatomines were collected in rural communities and analyzed for blood feeding sources, parasite diversity and gut microbiota composition through a metagenomic and deep sequencing approach. Triatoma dimidiata predominated, followed by Rhodnius prolixus, Triatoma maculata, Rhodnius pallescens, Panstrongylus geniculatus and Eratyrus cuspidatus. Twenty-two species were identified as blood sources, resulting in an integrated transmission network with extensive connectivity among sylvatic and domestic host species. Only TcI parasites were detected, predominantly from TcIb but TcIa was also reported. The close relatedness of T. cruzi strains further supported the lack of separate transmission cycles according to habitats or triatomine species. Triatomine microbiota varied according to species, developmental stage and T. cruzi infection. Bacterial families correlated with the presence/absence of T. cruzi were identified. In conclusion, we identified a domestic transmission cycle encompassing multiple vector species and tightly connected with sylvatic hosts in the SNSM region, rather than an isolated domestic transmission cycle. Therefore, integrated interventions targeting all vector species and their contact with humans should be considered.https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000318507https://orcid.org/0000-0002-8093-0544https://scienti.minciencias.gov.co/gruplac/jsp/visualiza/visualizagr.jsp?nro=00000000008981jose.usmec@campusucc.edu.cohttps://scholar.google.com.co/citations?user=cU2KyT4AAAAJ&hl=e