Molecular characterization of mosquitoes (Diptera: Culicidae) from the Colombian rainforest

A few studies have carried out the taxonomic and molecular characterization of sylvatic mosquito species in Latin America, where some species have been incriminated as vectors for arboviruses and parasites transmission. The present study reports the molecular characterization of mosquito species in the Sierra Nevada de Santa Marta, a natural ecosystem in the Northern coast of Colombia. Manual capture methods were used to collect mosquitoes, and the specimens were identified via classical taxonomy. The COI marker was used for species confirmation, and phylogenetic analysis was performed using the neighbor-joining method, with the Kimura-2-Parameters model. Aedes serratus , Psorophora ferox , Johnbelkinia ulopus , Sabethes chloropterus , Sabethes cyaneus , Wyeomyia aporonoma , Wyeomyia pseudopecten , Wyeomyia ulocoma and Wyeomyia luteoventralis were identified. We assessed the genetic variability of mosquitoes in this area and phylogenetic reconstructions allowed the identification at the species level. Classical and molecular taxonomy demonstrated to be useful and complementary when morphological characteristics are not well preserved, or the taxonomic group is not represented in public molecular databases

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

Identificación de sujetos a riesgo de deficiencia de hierromediante el Indicé Receptor Soluble de Transferrina-Log Ferritina sérica en hombres afrodescendientes residentes en San Basilio de Palenque y Cartagena de Indias, DTyC., Bolívar, Colombia.

En el ámbito mundial, sin distingo de raza, edad, género ni procedencia geográfica, la anemia de mayor prevalencia es la anemia ferropénica. De la misma forma, son insuficientes los reportes nacionales e internacionales sobre la prevalencia de los estados subclínicos de hierro, especialmente en comunidad afrodescendiente. El objetivo de la investigación fue caracterizar una población masculina afrodescendiente de 73 hombres sanos con edades comprendidas entre los 16 y 30 años mediante el comportamiento del índice (sTfR- Log F.S.), residentes en San Basilio de Palenque y en Cartagena de Indias DTyC. Se determinaron los índices primarios eritroides: hemoglobina (Hb), hematocrito (Hto), estudio de sangre periférica (E.S.P.); el receptor soluble de transferrina (sTfR), la ferritina sérica (F.S.) y el índice receptor soluble de transferrina – Logaritmo de la ferritina sérica (sTfR-Log F.S.) El análisis estadístico se realizó mediante el software de SPSS versión 17,0.   En la población de San Basilio de Palenque el hallazgo hematológico por el algoritmo de mayor frecuencia se asoció a la enfermedad crónica acompañada de deficiencia de hierro en el 41.6%, seguido de deficiencia subclínica de hierro estadio II con un 33.3%. En Cartagena el hallazgo de mayor frecuencia fue la enfermedad crónica con deficiencia de hierro en 49%, seguido de deficiencia subclínica de hierro estadio II 20.41%. Las dos poblaciones evidenciaron un comportamiento hematológico similar en las diferentes variables, constituyéndose estos resultados en pioneros para futuras investigaciones de los estadios subclínicos que anteceden la deficiencia de hierro en afrodescendientes colombianos

Análisis filogenético del virus del chikungunya en Colombia: evidencia de selección purificadora en el gen E1

Introduction: Chikungunya virus (CHIKV) is a single-stranded positive sense RNA virus that belongs to the Alphavirus genus of the family Togaviridae. Its genome is 11.8 kb in length, and three genotypes have been identified worldwide: Asian, East/Central/South African (ECSA) and West African. Chikungunya fever is an acute febrile disease transmitted by Aedes spp. that usually presents with polyarthralgia and cutaneous eruption. Following introduction of the virus to the Americas in 2013, the first cases in Colombia occurred in September of 2014, and they reached a cumulative total of 399,932 cases by June of 2015. Objective: To identify the genotype or genotypes responsible for the current epidemic in Colombia and to describe the genetic variability of the virus in the country. Materials and methods: Serum samples from patients presenting with symptoms compatible with Chikungunya fever during 2014-2015 were selected for the study. RT-PCR products of the E1 gene from these samples were used for sequencing and subsequent phylogenetic and adaptive evolution analyses. Results: The study identified only the presence of the Asian genotype in Colombia. Comparing the Colombian sequences with other sequences from the Americas revealed an average of 0.001 base substitutions per site, with 99.7% and 99.9% nucleotide identity and 99.9% amino acid identity. The adaptive evolution analysis indicated that the E1 gene is under strong purifying selection. Conclusions: The first epidemic of Chikunguya fever in Colombia was caused by the circulation of the virus Asian genotype. Further genotypic surveillance of the virus in Colombia is required to detect possible changes in its epidemiology, fitness and pathogenicity.Introducción. El virus del chikungunya, perteneciente al género Alphavirus de la familia Togaviridae, es un virus ARN de 11,8 kb, de cadena sencilla y polaridad positiva, transmitido por Aedes spp. Se han identificado tres genotipos a nivel mundial: el de Asia, el del este-centro-sur de África (East/Central/South African, ECSA) y el de África occidental (West African, WA). La fiebre del chikungunya es una enfermedad febril aguda, acompañada principalmente de inflamación en las articulaciones y erupción cutánea. Después de su aparición en las Américas en el 2013, los primeros casos en Colombia ocurrieron en septiembre de 2014 y hasta junio del 2015 se habían notificado 399.932 casos.Objetivo. Identificar el genotipo o los genotipos responsables de la primera epidemia por el virus del chikungunya en Colombia y la variabilidad genética asociada a su dispersión en el territorio nacional.Materiales y métodos. Se seleccionaron muestras de suero de pacientes con síntomas indicativos de fiebre del chikungunya durante 2014 y 2015. Se hizo una transcripción inversa seguida de reacción en cadena de la polimerasa del gen E1, así como su secuenciación, análisis filogenético y análisis de evolución adaptativa.Resultados. Se demostró la presencia exclusiva del genotipo de Asia en Colombia. Se registró un promedio de 0,001 sustituciones de bases por sitio, una identidad de 99,7 a 99,9 % en los nucleótidos y de 99,9 % en los aminoácidos entre las secuencias colombianas y las secuencias de las Américas. Los análisis de evolución adaptativa indicaron una fuerte selección purificadora en el gen E1.Conclusiones. Se determinó la circulación del genotipo de Asia del virus del chikungunya como la causa de la primera epidemia en Colombia. Es necesario continuar con la vigilancia de genotipos, con el fin de detectar posibles cambios en la epidemiología, la eficacia (fitness) viral y la patogenia del virus

Genomic epidemiology supports multiple introductions and cryptic transmission of Zika virus in Colombia

BACKGROUND: Colombia was the second most affected country during the American Zika virus (ZIKV) epidemic, with over 109,000 reported cases. Despite the scale of the outbreak, limited genomic sequence data were available from Colombia. We sought to sequence additional samples and use genomic epidemiology to describe ZIKV dynamics in Colombia. METHODS: We sequenced ZIKV genomes directly from clinical diagnostic specimens and infected Aedes aegypti samples selected to cover the temporal and geographic breadth of the Colombian outbreak. We performed phylogeographic analysis of these genomes, along with other publicly-available ZIKV genomes from the Americas, to estimate the frequency and timing of ZIKV introductions to Colombia. RESULTS: We attempted PCR amplification on 184 samples; 19 samples amplified sufficiently to perform sequencing. Of these, 8 samples yielded sequences with at least 50% coverage. Our phylogeographic reconstruction indicates two separate introductions of ZIKV to Colombia, one of which was previously unrecognized. We find that ZIKV was first introduced to Colombia in February 2015 (95%CI: Jan 2015 - Apr 2015), corresponding to 5 to 8 months of cryptic ZIKV transmission prior to confirmation in September 2015. Despite the presence of multiple introductions, we find that the majority of Colombian ZIKV diversity descends from a single introduction. We find evidence for movement of ZIKV from Colombia into bordering countries, including Peru, Ecuador, Panama, and Venezuela. CONCLUSIONS: Similarly to genomic epidemiological studies of ZIKV dynamics in other countries, we find that ZIKV circulated cryptically in Colombia. More accurately dating when ZIKV was circulating refines our definition of the population at risk. Additionally, our finding that the majority of ZIKV transmission within Colombia was attributable to transmission between individuals, rather than repeated travel-related importations, indicates that improved detection and control might have succeeded in limiting the scale of the outbreak within Colombia

Nuclei ultrastructural changes of C6/36 cells infected with virus dengue type 2

Introduction: Dengue virus replication has been considered mainly cytoplasmic, however, studies indicate that some flaviviruses may use the intranuclear pathway as part of the machinery that the virus uses to increase infection capacity in the host cell. This paper describes alterations at nuclear level in the cell infected with dengue, which are likely involved in the virus replication processes. Objective: This paper addresses the ultrastructural observations of C6/36 cells of the Aedes albopictus mosquito infected with dengue virus type 2. Materials and methods: C6/36 cells were infected in culture medium with the serum of a patient positively diagnosed for dengue 2. Subsequently, the cells were incubated for 10 days and the cytopathic effect was assessed. The cells were processed for immunofluorescence assays and transmission electron microscopy. Results: The immunofluorescence assays confirmed the presence of viral protein E associated with cellular syncytia in the culture. In the ultrastructural study, the infected cells showed vesicular-tubular structures and dilated cisterns of the endoplasmic reticulum at the cytoplasmic level. Viral particles were found exclusively in cytoplasm localized within the vacuoles. Nuclei of cellular syncytia showed membrane structures arranged in a circular shape and, in some cases, these syncytia displayed lysis; in no case viral particles were observed at the nuclear level. Conclusions: The ultrastructural alterations of nuclei in cells infected with the dengue virus using electron microscopy techniques had not been reported before, as far as we know. It is likely that such modifications are associated with replicative processes at an intranuclear level as an alternate replication mechanism

Análisis de determinantes genéticos de virulencia en cepas del virus dengue tipo 2

El virus dengue (DENV) es el agente etiológico del dengue, una de las enfermedades más importantes transmitidas por vector y es según la Organización Mundial de la Salud (OMS). Anualmente DENV afecta 50 - 200 millones de personas en el mundo y en Colombia se estima que casi 24 millones son susceptibles a la infección con el virus. Durante el periodo 2013 – 2015 se notificaron 669,631 casos de dengue en el país. Clínicamente, la enfermedad se clasifica como dengue (con y sin signos de alarma) y dengue grave el cual puede ser fatal. Aunque el principal factor asociado a la severidad es la ocurrencia de infecciones secundarias por serotipos heterólogos llevando a una potenciación mediada por anticuerpos, un número creciente de estudios sugiere la existencia de determinantes genéticos de virulencia en diferentes regiones del genoma viral y asociados a la severidad de la enfermedad. El objetivo fue identificar potenciales determinantes genéticos de virulencia asociados en cambios fenotípicos in vitro en cepas de DENV-2 y en el desarrollo de los diferentes cuadros clínicos de la enfermedad. La estrategia metodológica desarrollada en esta investigación incluyó selección de muestras de suero serotipificadas como DENV-2 y de pacientes con dengue y dengue grave. Posteriormente, se realizó aislamiento viral, amplificación y secuenciación del gen de la envoltura y de la región 5’ UTR. Por medio de alineamiento de secuencias y análisis filogenético se determinó el genotipo viral y las relaciones evolutivas entre cepas. Se identificaron estructuras secundarias conservadas de ARN mediante el análisis in silico. Finalmente fueron observadas infecciones in vitro por medio de curvas de crecimiento a diferentes tiempos post-infección y tituladas mediante ensayos de placa. Todas las cepas analizadas pertenecen al genotipo Asiático/Americano y se ubicaron en dos linajes genéticos. Las mismas estructuras secundarias conservadas estuvieron presentes en todas las cepas. En las regiones genómicas analizadas no se identificaron potenciales determinantes genéticos de virulencia y la caracterización in vitro no revela que existan diferencias significativas entre las cepas asociadas a los diferentes cuadros clínicos. Se concluye que no es posible establecer ninguna asociación entre determinantes genéticos de virulencia con las características in vitro de las cepas, ni con los distintos cuadros clínicos de la enfermedad, aunque se identifican posibles estructuras secundarias con algún tipo de funcionalidad potencial no conocido hasta el momento.Abstract. The Dengue Virus (DENV) is the Etiologic Agent of dengue, one of the most important vector-borne diseases, according to the World Health Organization (OMS). Annually the virus affects around 50-200 million people in the world and in Colombia it is estimated that nearly 24 million are susceptible to infection with the virus. During the period 2013 - 2015 669,631 cases of dengue fever were reported in the country. Clinically, the disease can be classified as dengue (with and without warning signs) and severe dengue which can be fatal. Although the main factor associated with the severity is the occurrence of secondary infections by heterologous serotypes leading to antibody-mediated enhancement (ADE), a growing number of studies suggest the existence of genetic determinants of virulence in different regions of the viral genome of DENV, possibly associated with the development of different clinical presentation. The objective of the research was to identify potential determinants of genetic virulence associated with phenotypic changes in vitro in strains of DENV-2 and the development of different clinical presentation of the disease. The methodological strategy developed in this research includes selection of serum samples serotyped as DENV-2 and patients with dengue and severe dengue. Subsequently, viral isolation, amplification and sequencing of the 5'UTR and envelope gene was performed. Through sequence alignment and phylogenetic analysis on viral genotype and the evolutionary relationships among strains it was determined. Conserved RNA secondary structures using in silico analysis identified. We were finally observed in vitro infections through growth curves at different times post-infection and titrated by plaque assays. All the strains tested belong to the Asian/American genotype and were placed into two genetic lineages. The same conserved secondary structures were present in all strains. In the genomic regions analyzed potential genetic determinants of virulence were not identified and in vitro characterization does not reveal any significant differences between strains associated with different clinical presentations. In conclusion, is not possible to establish any association between genetic determinants of virulence with the phenotypic characteristics of the strains, with different clinical presentations of the disease, even though possible secondary structures are identified with some kind of potential functionality not known until now.Maestrí

Zoonotic vaccinia in colombia: Cumulative evidence of the emergence of poxviruses in the world

The recent occurrence of vaccinia virus infections in humans and animals in Colombia, together with that reported for this and other species of the genus Orthopoxvirus in some South American, African, Asian and European countries, is supporting evidence of the emergence and re-emergence of the genus. This fact has become of great interest for public health around the world due to its biological and an epidemiological features, as was in the past the variola virus, one of its representatives. The emergence and re-emergence of the genus Orthopoxvirus may be a consequence of stopping vaccination against the variola virus in the 1970s and 1980s. This vaccination unsuspectedly induced cross-protective immunity to other species of that genus. This is a review of the history, biology and epidemiology of the main species of the genus Orthopoxvirus, together with its clinical presentation, social context and public health impact in the past, present and future

Secuenciación del SARS-CoV-2: la iniciativa tecnológica para fortalecer los sistemas de alerta temprana ante emergencias de salud pública en Latinoamérica y el Caribe

The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem on a scale unprecedented in the last 100 years, as has been the response focused on the rapid genomic characterization of SARS-CoV-2 in virtually all regions of the planet. This pandemic emerged during the era of genomic epidemiology, a science fueled by continued advances in next-generation sequencing. Since its recent appearance, genomic epidemiology included the precise identification of new lineages or species of pathogens and the reconstruction of their genetic variability in real time, evidenced in past outbreaks of influenza H1N1, MERS, and SARS. However, the global and uncontrolled scale of this pandemic created a scenario where genomic epidemiology was put into practice en masse, from the rapid identification of SARS-CoV-2 to the registration of new lineages and their active surveillance throughout the world. Prior to the COVID-19 pandemic, the availability of genomic data on circulating pathogens in several Latin America and the Caribbean countries was scarce or nil. With the arrival of SARS-CoV-2, this scenario changed significantly, although the amount of available information remains scarce and, in countries such as Colombia, Brazil, Argentina, and Chile, the genomic information of SARS-CoV-2 was obtained mainly by research groups in genomic epidemiology rather than the product of a public health surveillance policy or program. This indicates the need to establish public health policies aimed at implementing genomic epidemiology as a tool to strengthen surveillance and early warning systems against threats to public health in the region.La pandemia de COVID-19 causada por el SARS-CoV-2 es un problema de salud pública sin precedentes en los últimos 100 años, así como la respuesta centrada en la caracterización genómica del SARS-CoV-2 prácticamente en todas las regiones del planeta. Esta pandemia surgió durante la era de la epidemiología genómica impulsada por los continuos avances en la secuenciación de próxima generación. Desde su reciente aparición, la epidemiología genómica permitió la identificación precisa de nuevos linajes o especies de agentes patógenos y la reconstrucción de su variabilidad genética en tiempo real, lo que se hizo evidente en los brotes de influenza H1N1, MERS y SARS. Sin embargo, la escala global y descontrolada de esta pandemia ha generado una situación que obligó a utilizar de forma masiva herramientas de la epidemiología genómica como la rápida identificación del SARS-CoV-2 y el registro de nuevos linajes y su vigilancia activa en todo el mundo. Antes de la pandemia de COVID-19 la disponibilidad e datos genómicos de agentes patógenos circulantes en varios países de Latinoamérica y el Caribe era escasa o nula. Con la llegada del SARS-CoV-2 dicha situación cambió significativamente, aunque la cantidad de información disponible sigue siendo escasa y, en países como Colombia, Brasil, Argentina y Chile, la información genómica del SARS-CoV-2 provino principalmente de grupos de investigación en epidemiología genómica más que como producto de una política o programa de vigilancia en salud pública