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1.
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
2.
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
3.
Int J Antimicrob Agents ; 38(5): 435-41, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21840180

ABSTRACT

In the present work, the antiviral activity of brequinar (BQR) against the replication of Cantagalo virus was evaluated. BQR is a potent inhibitor of cellular dihydroorotate dehydrogenase, an enzyme of the de novo pyrimidine biosynthetic pathway. Infection in the presence of 0.5µM BQR reduced virus progeny production by >90%, revealing an EC(50) (drug concentration required to inhibit 50% of virus replication) of 0.017µM. Replication of other orthopoxviruses was also inhibited by BQR at similar levels. In the presence of the drug, virus early proteins accumulated to control levels, whereas late gene expression was severely impaired. This result was confirmed by indirect immunofluorescence assays and analysis of time-regulated expression of a reporter gene under the control of a virus promoter. Both assays revealed nearly 90% inhibition of late gene expression. BQR also blocked virus DNA replication, which accounted for the subsequent inhibition of virus late gene expression. The ablation of virus DNA replication, late gene expression and infectious progeny production was restored to control levels when infected cells were co-treated with uridine (URD) and BQR. These data demonstrated that BQR targeted virus DNA synthesis by depleting the cellular pyrimidine pool, which was bypassed by the salvage pathway when URD was added to the cell cultures.


Subject(s)
Antiviral Agents/pharmacology , Biphenyl Compounds/pharmacology , Enzyme Inhibitors/pharmacology , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Vaccinia virus/drug effects , Virus Replication/drug effects , Animals , Cell Line , DNA Replication/drug effects , Dihydroorotate Dehydrogenase , Gene Expression Regulation , Kidney/cytology , Kidney/virology , Pyrimidines/biosynthesis , Vaccinia virus/classification , Vaccinia virus/enzymology , Vaccinia virus/physiology , Viral Proteins/genetics , Viral Proteins/metabolism
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