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Las infecciones con el virus del dengue, transmitidas al humano por mosquitos del género Aedes sp., constituyen un problema de salud pública para las regiones tropicales y subtropicales del planeta. El ciclo de replicación del virus del dengue se lleva a cabo principalmente en el citoplasma de las células, en estrecha asociación con organelos membranosos como el retículo endoplasmático rugoso y el complejo de Golgi. En esta revisión se analiza la respuesta al estrés de estos organelos, ante la infección viral, tanto en células de vertebrados como de mosquitos, y se discuten las implicaciones de esta respuesta en la biología de estos agentes.

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. A total of 817 mosquito targets were identified as protein–protein interacting with DENV NS1. Approximately 14% of them coincide with interactomes previously obtained in vertebrate cells, including the oligosaccharide transferase complex, the chaperonin containing TCP-1, vesicle localization, and ribosomal proteins. Notably, other protein pathways not previously reported in vertebrate cells, such as epigenetic regulation and RNA silencing, were also found in the NS1 interactome in mosquito cells. Due to the novel and strong interactions observed for NS1 and the epigenetic regulator DIDO1 (Death-Inducer Obliterator 1), the role of DIDO1 in viral replication was further explored. Interactions between NS1 and DIDO1 were corroborated in infected mosquito cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected cells silenced for DIDO1 revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD, and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response necessary for flavivirus replication in mosquito cells. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication, and add to the understanding of NS1 as a multifunctional protein. IMPORTANCE Dengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as transcription and epigenetic regulation, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1 revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

Septins are a family of GTP-binding proteins identified in insects and mammals. Septins are components of the cytoskeleton and participate in cytokinesis, chromosomal segregation, intracellular vesicular traffic, and response to pathogens. Human septin 6 was identified as necessary for hepatitis C virus replication. Information about host factors necessary for flavivirus replication in mosquitoes is scarce. Thus, the role of septins in the replicative cycle of dengue virus in Aedes spp. derived cells was investigated. Through bioinformatic analysis, sequences of septin-like proteins were identified. Infected mosquito cells showed increased expression of Sep2. Colocalization analysis, proximity ligation and immunoprecipitation assays indicated that Sep2 interacts with proteins E, NS3 and NS5, but not NS1. Immunoelectron microscopy evidenced the presence of AalSep2 in replicative complexes. Finally, silencing of Sep2 expression resulted in a significant decrease in virus progeny, indicating that Sep2 is a host factor participating in dengue virus replication in mosquito cells.

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. Approximately 14% of the 817 identified proteins coincide with interactomes obtained in vertebrate cells, including ontology groups of the oligosaccharide transferase complex, the chaperonin containing TCP-1, and nuclear import and export, vesicle localization and ribosomal proteins. Notably, other protein pathways such as epigenetic regulation and RNA silencing, not previously reported in vertebrate cells, were also found in the NS1 interactome in mosquito cells. Due to the novel interaction observed for NS1 and DIDO1 (Death Inducer-Obliterator 1), we further explored the role of DIDO1 in viral replication. Interactions between NS1 and DIDO1were corroborated in infected C6/36 and Aag2 cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression in C6/36 and Aag2 cells results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected C6/36 silenced for DIDO1, revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response and necessary for flavivirus replication. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication and add to the understanding of NS1 as a multifunctional protein.ABSTRACT IMPORTANCEDengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as epigenetic regulation and RNA silencing, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in vertebrate and mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1, revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. Few examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on Golgi complex imposed by virion and NS1 processing and secretion.

Background & objectives: Trypanosoma cruzi and Leishmania spp. are protozoans that cause American trypanosomiasis and leishmaniasis, respectively. In endemic foci where both diseases coincide, coinfection can occur. The objective of this work was the characterization of the parasites involved in coinfection in several endemic areas of Venezuela. Methods: Molecular characterization was done in 30 samples of several species of mammals (Didelphis marsupialis, Equus mulus, Rattus rattus, Canis familiaris, Felis catus, and Sciurus granatensis) from the states of Anzoategui, Cojedes and Capital District diagnosed with T. cruzi and Leishmania spp. coinfections. For the typing of T. cruzi DTUs, the markers of miniexon, 24Sa rDNA, 18Sa rDNA, and hsp60-PCR-RFLP (EcoRV) were used. Infection by Leishmania spp. was characterized by miniexon multiplex PCR for complexes of Leishmania and ITS1-PCR-RFLP (HaeIII, HhaI, and RsaI) for the identification of the species. Results: The T. cruzi TcI was present in 100% of the coinfected mammals, which included 76.7% of triple infection by T. cruzi TcI-complex–L. (L) mexicana–L. infantum/chagasi, 13.3% of double infection by T. cruzi TcI-L. mexicana and 10% of double infection by T. cruzi Tcl—L. infantum/chagasi. Interpretation & conclusion: These results suggest that the double or triple infection is a phenomenon existing in almost all the coendemics areas and mammals studied, which might influence the mechanisms of adaptation and pathogenicity of these parasites.

Chlamydia psittaci (Cp) es una bacteria intracelular obligada transmitida a través de aerosoles derivados de secreciones nasales y ópticas, tejidos, heces y plumas. Comúnmente es identificada en aves, sin embargo, han emergido genotipos capaces de infectar nuevos reservorios mamíferos. Por ello, se buscó ADN de Cp en muestras de cinco individuos de Mustella putorios furo y un hisopado cloacal de un individuo de Colinus cristatus en cautiverio en Venezuela a través de la PCR-anidada, amplificando un segmento del gen 16S ADNr. Se demostró la presencia de Cp en un Colinus cristatus con signos de clamidiosis y en cuatro Mustella putorios furo sin signos clínicos de clamidiosis. Se indica un posible nuevo reservorio para Cp, donde el contacto con productos de excreción de Colinus cristatus con manifestaciones clínicas de clamidiosis, hacinamiento, inadecuada ventilación, contacto con productos de excreción de Psittaciformes y condiciones sanitarias deficientes favoreció la infección por Cp. Se desconoce el total de reservorios de Cp, por ello la notificación de los aislados permite el entendimiento, distribución y diversidad de agentes clamidiales en fauna silvestre y en cautiverio.

Objetivos. Comparar dos protocolos de extracción de ADN de Trypanosoma cruzi para su uso en la amplificación de ADN de minicírculos de kinetoplasto (ADNk) mediante la técnica de Reacción en Cadena de Polimerasa (PCR). Materiales y métodos. Se cultivaron epimastigotas de T. cruzi en condiciones exénicas obteniéndose masas entre 1,5 hasta 100 x 10(6) parásitos. A partir de estas se procedió a la extracción de ADN mediante dos protocolos: extracción con solventes orgánicos (fenol/cloroformo), y empleo de resina (Chelex®100), a partir de los diferentes sedimentos parasitarios. La concentración y pureza del ADN se determinó por espectrofotometría y la integridad se evaluó mediante electroforesis en geles de agarosa. Se realizó el análisis de varianza y comparaciones de medias mediante la prueba de Tukey, utilizando el software Statistix 8.0. Resultados. Se realizaron diez extracciones de ADN de cada una de las diferentes cantidades de parásitos sedimentados. En la extracción de ADN con la resina Chelex®100 se obtuvo mayor rendimiento, pero menor pureza e integridad respecto a la extracción con solventes orgánicos. Sin embargo, permitió la amplificación del producto de 330 pb de ADNk de T. cruzi. Conclusiones. Aun cuando la técnica de Chelex®100 proporcionó menor pureza e integridad del ADN, permitió la amplificación con éxito de ADNk por PCR, evitando el uso de técnicas laboriosas y solventes orgánicos tóxicos Objectives. To compare two extraction protocols of Trypanosoma cruzi DNA for use in DNA amplification of kinetoplast minicircles (kDNA) through the technique of Polymerase Chain Reaction (PCR). Materials and methods. Epimastigotes of T. cruzi were cultured in axenic conditions and masses from 1.5 to 100 x 106 parasites were obtained. DNA extraction was performed using two protocols: extraction with organic solvents (phenol/chloroform), and with resin (Chelex®100), from different parasitic sediments. Concentration and purity of DNA was determined by spectrophotometry, and integrity was assessed by agarose gel electrophoresis. Analysis of variance and comparisons of means were performed through Tukey’s test, using the Statistix 8.0 software. Results. Ten DNA extractions were done of each one of the different amounts of parasitic sediments. In the DNA extraction with Chelex®100 resin, a higher performance was obtained but a lower purity and integrity compared to the extraction with organic solvents. However, it allowed a product amplification of 330 bp of T. cruzi kDNA. Conclusions. Although the technique of Chelex®100 provided less purity and integrity of DNA, it allowed a successful amplification of kDNA by PCR, avoiding the use of laborious techniques and toxic organic solvents