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1.
Viruses ; 14(4)2022 04 15.
Article in English | MEDLINE | ID: mdl-35458545

ABSTRACT

Virus replication frequently results in the accumulation, re-assortment and re-combination of mutations, which contributes to their rapid adaptation to environmental changes and often advances the emergence of new virus variants or species [...].


Subject(s)
Viruses , Mutation , Virus Replication/genetics , Viruses/genetics
2.
Viruses ; 14(2)2022 02 16.
Article in English | MEDLINE | ID: mdl-35215998

ABSTRACT

Viral infections by endemic, emerging, and reemerging viruses are constantly challenging public health systems and health policies all over the world [...].


Subject(s)
Developing Countries , Virus Diseases , Health Policy , Humans , Public Health
3.
Methods Mol Biol ; 2409: 207-222, 2022.
Article in English | MEDLINE | ID: mdl-34709644

ABSTRACT

This chapter will discuss reliable and relatively easy and fast strategies to evaluate the integrity of endothelial cell monolayers when infected by dengue virus (DENV). Human brain microvascular endothelial cells (HBMEC) were exploited here as general model of vessel wall core, but it may also be used as an in vitro simplified model of blood brain barrier (BBB). The integrity of endothelial cells monolayer can be inferred using a transwell culture system by: (1) measuring transendothelial electrical resistance (TEER) using a Voltohmmeter; (2) analyzing the monolayer permeability to fluorescent-conjugated proteins and fluorimetric assay; (3) investigating virus extravasation by quantitative RT-PCR and plaque conventional assay. The rational to use those strategies is that vascular alterations are often observed during dengue infection, being associated to disease severity. The vasculature core consists of a barrier of endothelial cells, which are tightly adhered by the expression of adhesion molecules and tight junctions. This structure must be preserved in order to control the flux of cells and metabolites from the circulation to the tissues and to maintain vascular homeostasis. Therefore, experimental assays that allow evaluation of endothelial integrity can be useful platforms to further understand disease pathogenesis and screen pharmaceutical interventions to control vascular disturbance.


Subject(s)
Endothelial Cells , Blood-Brain Barrier , Brain , Capillary Permeability , Cells, Cultured , Electric Impedance , Humans , Permeability
4.
Viruses ; 13(2)2021 02 06.
Article in English | MEDLINE | ID: mdl-33562062

ABSTRACT

This Special Issue of Viruses is a collection of the current knowledge on a broad range of emerging human, animal, and plant viral diseases [...].


Subject(s)
Communicable Diseases, Emerging/epidemiology , Communicable Diseases, Emerging/transmission , Virus Diseases/epidemiology , Virus Diseases/transmission , Viruses/classification , Animals , Humans , Plant Diseases/virology , Plants/virology
5.
Viruses ; 12(3)2020 03 11.
Article in English | MEDLINE | ID: mdl-32168932

ABSTRACT

Emerging viruses represent a major concern for public health offices. Climate changes, the international migration of people and products, deforestation, and other anthropogenic activities (and their consequences) have been historically and continuously related to the emerging and re-emerging of new viruses, triggering an increasing number of notified outbreaks, epidemics, and pandemics. [...].


Subject(s)
Communicable Diseases, Emerging/epidemiology , Communicable Diseases, Emerging/prevention & control , Virus Diseases/epidemiology , Virus Diseases/prevention & control , Animals , Biological Evolution , Communicable Diseases, Emerging/virology , Humans , Public Health , Public Health Surveillance , Virus Diseases/virology
6.
PLoS Negl Trop Dis ; 13(6): e0007500, 2019 06.
Article in English | MEDLINE | ID: mdl-31216268

ABSTRACT

BACKGROUND: Leishmania parasites are transmitted to vertebrate hosts by phlebotomine sandflies and, in humans, may cause tegumentary or visceral leishmaniasis. The role of PKR (dsRNA activated kinase) and Toll-like receptor 3 (TLR3) activation in the control of Leishmania infection highlights the importance of the engagement of RNA sensors, which are usually involved in the antiviral cell response, in the fate of parasitism by Leishmania. We tested the hypothesis that Phlebovirus, a subgroup of the Bunyaviridae, transmitted by sandflies, would interfere with Leishmania infection. METHODOLOGY/PRINCIPAL FINDINGS: We tested two Phlebovirus isolates, Icoaraci and Pacui, from the rodents Nectomys sp. and Oryzomys sp., respectively, both natural sylvatic reservoir of Leishmania (Leishmania) amazonensis from the Amazon region. Phlebovirus coinfection with L. (L.) amazonensis in murine macrophages led to increased intracellular growth of L. (L.) amazonensis. Further studies with Icoaraci coinfection revealed the requirement of the PKR/IFN1 axis on the exacerbation of the parasite infection. L. (L.) amazonensis and Phlebovirus coinfection potentiated PKR activation and synergistically induced the expression of IFNß and IL-10. Importantly, in vivo coinfection of C57BL/6 mice corroborated the in vitro data. The exacerbation effect of RNA virus on parasite infection may be specific because coinfection with dengue virus (DENV2) exerted the opposite effect on parasite load. CONCLUSIONS: Altogether, our data suggest that coinfections with specific RNA viruses shared by vectors or reservoirs of Leishmania may enhance and sustain the activation of host cellular RNA sensors, resulting in aggravation of the parasite infection. The present work highlights new perspectives for the investigation of antiviral pathways as important modulators of protozoan infections.


Subject(s)
Bunyaviridae Infections/complications , Coinfection/immunology , Disease Susceptibility , Interferon-beta/metabolism , Interleukin-10/metabolism , Leishmaniasis/immunology , eIF-2 Kinase/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Leishmania/immunology , Mice, Inbred C57BL , Models, Theoretical , Phlebovirus/immunology
7.
Front Microbiol ; 10: 525, 2019.
Article in English | MEDLINE | ID: mdl-30984122

ABSTRACT

Human infection by different flaviviruses may cause severe neurologic syndromes, through pathogenic mechanisms that are still largely unknown. Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), yellow fever virus (YFV), dengue virus (DENV), and tick-borne encephalitis virus (TBEV) are believed to reach the central nervous system by a hematogenous route, upon crossing the blood-brain barrier. Although the disruption of BBB during flavivirus infection has been largely evidenced in experimental models, the relevance of BBB breakdown for virus entering the brain was not completely elucidated. In vitro models of BBB had demonstrated that these viruses replicated in brain microvascular endothelial cells (BMECs), which induced downregulation of tight junction proteins and increased the permeability of the barrier. Other reports demonstrated that infection of BMECs allowed the basolateral release of infectious particles, without a remarkable cytopathic effect, what might be sufficient for virus invasion. Virus replication and activation of other cells associated to the BBB, mostly astrocytes and microglia, were also reported to affect the endothelial barrier permeability. This event might occur simultaneously or after BMECs infection, being a secondary effect leading to BBB disruption. Importantly, activation of BMECs, astrocytes, and microglia by flaviviruses was associated to the expression and secretion of inflammatory mediators, which are believed to recruit leukocytes to the CNS. The leukocyte infiltrate could further mediate viral invasion through a Trojan horse mechanism and might contribute to BBB breakdown and to neurological alterations. This review discussed the previous studies regarding in vitro and in vivo models of JEV, WNV, ZIKV, YFV, DENV, and TBEV infection and addressed the pathways for BBB overcome and invasion of the CNS described for each virus infection, aiming to increment the knowledge and stimulate further discussion about the role of BBB in the neuropathogenesis of flavivirus infection.

8.
Arch Virol ; 163(10): 2687-2699, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29876782

ABSTRACT

Zika virus (ZIKV) is a flavivirus that has been highly correlated with the development of neurological disorders and other malformations in newborns and stillborn fetuses after congenital infection. This association is supported by the presence of ZIKV in the fetal brain and amniotic fluid, and findings suggest that infection of the placental barrier is a critical step for fetal ZIKV infection in utero. Therefore, relevant models to investigate the interaction between ZIKV and placental tissues are essential for understanding the pathogenesis of Zika syndrome. In this report, we demonstrate that explant tissue from full-term human placentas sustains a productive ZIKV infection, though the results depend on the strain. Viral infection was found to be associated with pro-inflammatory cytokine expression and apoptosis of the infected tissue, and these findings confirm that placental explants are targets of ZIKV replication. We propose that human placental explants are useful as a model for studying ZIKV infection ex vivo.


Subject(s)
Apoptosis/immunology , Placenta/virology , Zika Virus Infection/pathology , Zika Virus/immunology , Animals , Cell Line , Chlorocebus aethiops , Cytokines/biosynthesis , Cytokines/immunology , Female , Humans , Infant, Newborn , Inflammation/immunology , Placenta/pathology , Pregnancy , Vero Cells , Viral Load , Virus Replication/physiology , Zika Virus/growth & development
9.
J Virol Methods ; 246: 65-74, 2017 08.
Article in English | MEDLINE | ID: mdl-28445704

ABSTRACT

The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols. ZIKV produces a productive infection in human, non-human primate, and rodents-derived cell lines, with different efficacies. The highest yield of ZIKV-AFR and ZIKV-BR infectious progeny was obtained at 7days post infection in C6/36 cells (7×107 and 2×108 PFU/ml, respectively). However, high titers of ZIKV-AFR could be obtained at earlier time points in Vero cells (2.5×107PFU/ml at 72hpi), whereas ZIKV-BR titers reached 108 PFU/ml at 4dpi in C6/36 cells. High yield of purified virus was obtained by purification through a discontinuous sucrose gradient. This optimized procedure will certainly contribute to future studies of virus structure and vaccine development. Beyond the achievement of efficient virus propagation, the normalization of these protocols will also allow different laboratories around the world to better compare and discuss data regarding different features of ZIKV biology and disease, contributing to more efficient collaborations and progression in ZIKV research.


Subject(s)
Virology/standards , Virus Cultivation/standards , Virus Replication , Zika Virus/growth & development , Zika Virus/isolation & purification , Animals , Brain/cytology , Cell Line , Centrifugation , Chlorocebus aethiops , Culicidae/cytology , Endothelial Cells/virology , Genome, Viral , Humans , Metagenomics , Vero Cells , Viral Load/methods , Virology/methods , Zika Virus/genetics
10.
PLoS One ; 9(6): e99887, 2014.
Article in English | MEDLINE | ID: mdl-24932692

ABSTRACT

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.


Subject(s)
Endosomes/metabolism , Lysosomal-Associated Membrane Protein 1/chemistry , Lysosomal-Associated Membrane Protein 1/metabolism , Lysosomes/metabolism , Secretory Pathway , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Exosomes/metabolism , Female , HEK293 Cells , Humans , Immunization , Lymphocyte Activation/immunology , Mice, Inbred BALB C , Protein Structure, Tertiary , Proteolysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Structure-Activity Relationship , Transcription, Genetic , gag Gene Products, Human Immunodeficiency Virus/metabolism
11.
Virology ; 435(2): 281-92, 2013 Jan 20.
Article in English | MEDLINE | ID: mdl-23089253

ABSTRACT

Dengue virus (DENV) infection is associated to exacerbated inflammatory response and structural and functional alterations in the vascular endothelium. However, the mechanisms underlying DENV-induced endothelial cell activation and their role in the inflammatory response were not investigated so far. We demonstrated that human brain microvascular endothelial cells (HBMECs) are susceptible to DENV infection, which induces the expression of the cytoplasmic pattern recognition receptor (PRR) RIG-I. Infection of HBMECs promoted an increase in the production of type I IFN and proinflammatory cytokines, which were abolished after RIG-I silencing. DENV-infected HBMECs also presented a higher ICAM-1 expression dependent on RIG-I activation as well. On the other hand, ablation of RIG-I did not interfere with virus replication. Our data suggest that RIG-I activation by DENV may participate in the disease pathogenesis through the modulation of cytokine release and expression of adhesion molecules, probably contributing to leukocyte recruitment and amplification of the inflammatory response.


Subject(s)
DEAD-box RNA Helicases/metabolism , Dengue Virus/pathogenicity , Endothelial Cells/immunology , Endothelial Cells/virology , Up-Regulation , Animals , Brain/blood supply , Cell Line , Cytokines/metabolism , DEAD Box Protein 58 , Dengue/immunology , Dengue/virology , Endothelium, Vascular/cytology , Humans , Intercellular Adhesion Molecule-1 , Interferon-beta/biosynthesis , Microcirculation , Receptors, Immunologic
12.
PLoS Pathog ; 3(11): e185, 2007 Nov.
Article in English | MEDLINE | ID: mdl-18052532

ABSTRACT

Although the concept that dendritic cells (DCs) recognize pathogens through the engagement of Toll-like receptors is widely accepted, we recently suggested that immature DCs might sense kinin-releasing strains of Trypanosoma cruzi through the triggering of G-protein-coupled bradykinin B2 receptors (B2R). Here we report that C57BL/6.B2R-/- mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates as compared to B2R+/+ mice. qRT-PCR revealed a 5-fold increase in T. cruzi DNA (14 d post-infection [p.i.]) in B2R-/- heart, while spleen parasitism was negligible in both mice strains. Analysis of recall responses (14 d p.i.) showed high and comparable frequencies of IFN-gamma-producing CD4+ and CD8+ T cells in the spleen of B2R-/- and wild-type mice. However, production of IFN-gamma by effector T cells isolated from B2R-/- heart was significantly reduced as compared with wild-type mice. As the infection continued, wild-type mice presented IFN-gamma-producing (CD4+CD44+ and CD8+CD44+) T cells both in the spleen and heart while B2R-/- mice showed negligible frequencies of such activated T cells. Furthermore, the collapse of type-1 immune responses in B2R-/- mice was linked to upregulated secretion of IL-17 and TNF-alpha by antigen-responsive CD4+ T cells. In vitro analysis of tissue culture trypomastigote interaction with splenic CD11c+ DCs indicated that DC maturation (IL-12, CD40, and CD86) is controlled by the kinin/B2R pathway. Further, systemic injection of trypomastigotes induced IL-12 production by CD11c+ DCs isolated from B2R+/+ spleen, but not by DCs from B2R-/- mice. Notably, adoptive transfer of B2R+/+ CD11c+ DCs (intravenously) into B2R-/- mice rendered them resistant to acute challenge, rescued development of type-1 immunity, and repressed TH17 responses. Collectively, our results demonstrate that activation of B2R, a DC sensor of endogenous maturation signals, is critically required for development of acquired resistance to T. cruzi infection.


Subject(s)
Chagas Disease/immunology , Dendritic Cells/immunology , Kinins/metabolism , Receptor, Bradykinin B2/immunology , Th1 Cells/immunology , Adoptive Transfer , Animals , Chagas Disease/metabolism , Dendritic Cells/metabolism , Flow Cytometry , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Interleukin-17/biosynthesis , Kinins/immunology , Lymphocyte Activation/immunology , Mice , Mice, Mutant Strains , Polymerase Chain Reaction , Receptor, Bradykinin B2/metabolism , Th1 Cells/metabolism , Trypanosoma cruzi , Tumor Necrosis Factor-alpha/biosynthesis
13.
Immunology ; 112(1): 126-33, 2004 May.
Article in English | MEDLINE | ID: mdl-15129672

ABSTRACT

Antigen presentation by major histocompatibility complex type II (MHC II) molecules and activation of CD4+ helper T cells are critical for the generation of immunological memory. We previously described a DNA vaccine encoding human immunodeficiency virus-1 p55Gag as a chimera with the lysosome-associated membrane protein (LAMP/gag). The LAMP/gag chimera protein traffics to the MHC II compartment of transfected cells and elicits enhanced immune responses as compared to a DNA vaccine encoding native gag not targeted to the MHC II compartment. We have now investigated the long-term responses of immunized mice and show that the LAMP/gag DNA vaccine promotes long-lasting B cell- and CD4+ and CD8+ T-cell memory responses induced by DNA encoding non-targeted Gag decay rapidly and elicit very low or undetectable levels of gag DNA is sufficient to generate T-cell memory. Following this initial priming immunization with LAMP/gag DNA, booster immunizations with native gag DNA or the LAMP/gag chimera are equally efficient in eliciting B- and T-cell secondary responses, results in accordance with observations that secondary expansion of CD8+ cells in the boost phase does not require additional CD4+ help. These findings underscore the significance of targeting DNA-encoded vaccine antigens to the MHC II processing compartments for induction of long-term immunological memory.


Subject(s)
AIDS Vaccines/immunology , Antigens, CD/immunology , Gene Products, gag/immunology , HIV-1/immunology , Vaccines, DNA/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Female , HIV Antibodies/biosynthesis , Histocompatibility Antigens Class II/immunology , Immunologic Memory , Lymphocyte Activation/immunology , Lysosomal Membrane Proteins , Mice , Mice, Inbred BALB C
14.
J Biol Chem ; 278(39): 37926-36, 2003 Sep 26.
Article in English | MEDLINE | ID: mdl-12824194

ABSTRACT

Several genetic vaccines encoding antigen chimeras containing the lysosome-associated membrane protein (LAMP) translocon, transmembrane, and cytoplasmic domain sequences have elicited strong mouse antigen-specific immune responses. The increased immune response is attributed to trafficking of the antigen chimera to the major histocompatibility class II (MHC II) compartment where LAMP is colocalized with MHC II. In this report, we describe a new form of an HIV-1 p55gag DNA vaccine, with the gag sequence incorporated into the complete LAMP cDNA sequence. Gag encoded with the translocon, transmembrane and cytoplasmic lysosomal membrane targeting sequences of LAMP, without the luminal domain, was poorly expressed, did not traffic to lysosomes or MHC II compartments of transfected cells, and elicited a limited immune response from DNA immunized mice. In contrast, addition of the LAMP luminal domain sequence to the construct resulted in a high level of expression of the LAMP/Gag protein chimera in transfected cells that was further increased by including the inverted terminal repeat sequences of the adeno-associated virus to the plasmid vector. This LAMP/Gag chimera with the complete LAMP protein colocalized with endogenous MHC II of transfected cells and elicited strong cellular and humoral immune responses of immunized mice as compared with the response to DNA-encoding native Gag, with a 10-fold increase in CD4+ responses, a 4- to 5-fold increase in CD8+ T-cell responses, and antibody titers of >100,000. These results reveal novel roles of the LAMP luminal domain as a determinant of Gag protein expression, lysosomal trafficking, and possibly of the immune response to Gag.


Subject(s)
AIDS Vaccines/immunology , Cell Adhesion Molecules, Neuronal/immunology , Gene Products, gag/immunology , HIV-1/immunology , Histocompatibility Antigens Class II/physiology , Protein Precursors/immunology , Recombinant Fusion Proteins/immunology , Vaccines, DNA/immunology , 3T3 Cells , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COS Cells , Cell Adhesion Molecules, Neuronal/genetics , Dose-Response Relationship, Immunologic , GPI-Linked Proteins , Gene Products, gag/genetics , Humans , Immunization , Lysosomes/metabolism , Mice , Plasmids , Protein Precursors/genetics , Protein Transport
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