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Despite many advances on the understanding of dengue pathogenesis in the last decades, some questions remained to be clarified. The virulence of the pathogen and the host immune response are the main factors involved in pathogenesis of dengue infection. In addition, skin dendritic cells (DCs) are one of the primary targets for dengue virus infection. After infection, DCs process and present antigens to T cells and also secrete cytokines that shape the immune response. Although relevant for the development of antiviral immune response, an imbalance in the cytokine production by immune cells could lead to cytokine storm observed in severe dengue fever cases. Therefore, this chapter will describe the protocols for the in vitro differentiation of human monocytes into human monocyte-derived dendritic cells (mdDCs), followed by dengue virus infection, as well as the cytokine quantification produced by mdDCs using a cytometric bead array method.

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The Dengue pathophysiology has had several aspects determined over the years. However, some points remain elusive, such as the metabolic factors that regulate the massive B cell differentiation into antibody-secreting cells observed in Dengue patients. In this chapter, we describe an in vitro method capable of mimicking this Dengue-induced cell expansion. More specifically, this approach allows dengue virus-stimulated peripheral blood mononuclear cells (PBMCs) from healthy individuals to enhance the frequency of phenotypical and functional antibody-secreting cells (ASCs) after 7 days of culture. A manuscript recently published by Bonezi and colleagues displays results generated through this methodology.

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Zika virus (ZIKV) is an arthropod-born virus that is mainly transmitted to humans by mosquitoes of the genus Aedes spp. Since its first isolation in 1947, only a few human cases had been described until large outbreaks occurred on Yap Island (2007), French Polynesia (2013), and Brazil (2015). Most ZIKV-infected individuals are asymptomatic or present with a self-limiting disease and nonspecific symptoms such as fever, myalgia, and headache. However, in French Polynesia and Brazil, ZIKV outbreaks led to the diagnosis of congenital malformations and microcephaly in newborns and Guillain-Barré syndrome (GBS) in adults. These new clinical presentations raised concern from public health authorities and highlighted the need for anti-Zika treatments and vaccines to control the neurological damage caused by the virus. Despite many efforts in the search for an effective treatment, neither vaccines nor antiviral drugs have become available to control ZIKV infection and/or replication. Flavonoids, a class of natural compounds that are well-known for possessing several biological properties, have shown activity against different viruses. Additionally, the use of flavonoids in some countries as food supplements indicates that these molecules are nontoxic to humans. Thus, here, we summarize knowledge on the use of flavonoids as a source of anti-ZIKV molecules and discuss the gaps and challenges in this area before these compounds can be considered for further preclinical and clinical trials.

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Leydig cells play pivotal roles in eliciting male characteristics by producing testosterone and any damage to these cells can compromise male fertility Toxoplasma gondii (T. gondii) is an intracellular parasite capable to invade any nucleated cell, including cells from male reproductive system. Herein, we evaluated the capacity of RH strain of T. gondii to infect TM3 Leydig cells and the impact of this infection on testosterone and inflammatory mediators production. We first, by performing adherence, infection, and intracellular proliferation assays, we found a significant increase in the number of infected Leydig cells, peaking 48 h after the infection with T. gondii. Supernatants of TM3 infected cells exhibited, in a time-dependent manner, increased levels of testosterone as well as monocyte chemoattractant protein-1 (MCP-1) and interferon-γ (IFN-γ), which is correlated with the robust T. gondii infection. In conclusion, our study provides new insights regarding the harmful effects of T. gondii infection on male reproductive system.

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Genomic and epidemiological surveillance are paramount for the discovery of new viruses with the potential to cross species barriers. Here, we present a new member of the genus Alphavirus found in Trichoprosopon and Wyeomia mosquitoes, tentatively named Pirahy virus (PIRAV). PIRAV was isolated from mosquito pools collected in a rural area of Piraí do Sul, South Brazil. In vitro assays revealed that PIRAV replicates and causes cytopathic effects in vertebrate cell lines such as Vero E6, SH-SY5Y, BHK-21 and UMNSAH/DF-1. Genomic signature analysis supports these results showing a dinucleotide and codon usage balance compatible with several hosts. Phylogenetic analyses placed PIRAV basal to the Venezuelan equine encephalitis complex. Genome analyses, electron microscopy, and biological characterization show findings that may alert for the emergence of a new arbovirus in South America.

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The Zika virus (ZIKV) is an arthropod-borne virus that belongs to the Flaviviridae family. The ZIKV infection is usually asymptomatic or is associated with mild clinical manifestations; however, increased numbers of cases of microcephaly and birth defects have been recently reported. To date, neither a vaccine nor an antiviral treatment has become available to control ZIKV replication. Among the natural compounds recognized for their medical properties, flavonoids, which can be found in fruits and vegetables, have been found to possess biological activity against a variety of viruses. Here, we demonstrate that the citrus flavanone naringenin (NAR) prevented ZIKV infection in human A549 cells in a concentration-dependent and ZIKV-lineage independent manner. NAR antiviral activity was also observed when primary human monocyte-derived dendritic cells were infected by ZIKV. NAR displayed its antiviral activity when the cells were treated after infection, suggesting that NAR acts on the viral replication or assembly of viral particles. Moreover, a molecular docking analysis suggests a potential interaction between NAR and the protease domain of the NS2B-NS3 protein of ZIKV which could explain the anti-ZIKV activity of NAR. Finally, the results support the potential of NAR as a suitable candidate molecule for developing anti-ZIKV treatments.

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BACKGROUND: Zika virus (ZIKV) infections reported in recent epidemics have been linked to clinical complications that had never been associated with ZIKV before. Adaptive mutations could have contributed to the successful emergence of ZIKV as a global health threat to a nonimmune population. However, the causal relationships between the ZIKV genetic determinants, the pathogenesis and the rapid spread in Latin America and in the Caribbean remain widely unknown. OBJECTIVES: The aim of this study was to characterise three ZIKV isolates obtained from patient samples during the 2015/2016 Brazilian epidemics. METHODS: The ZIKV genomes of these strains were completely sequenced and in vitro infection kinetics experiments were carried out in cell lines and human primary cells. FINDINGS: Eight nonsynonymous substitutions throughout the viral genome of the three Brazilian isolates were identified. Infection kinetics experiments were carried out with mammalian cell lines A549, Huh7.5, Vero E6 and human monocyte-derived dendritic cells (mdDCs) and insect cells (Aag2, C6/36 and AP61) and suggest that some of these mutations might be associated with distinct viral fitness. The clinical isolates also presented differences in their infectivity rates when compared to the well-established ZIKV strains (MR766 and PE243), especially in their abilities to infect mammalian cells. MAIN CONCLUSIONS: Genomic analysis of three recent ZIKV isolates revealed some nonsynonymous substitutions, which could have an impact on the viral fitness in mammalian and insect cells.

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American cutaneous leishmaniasis is a zoonotic disease caused by protozoans of the genus Leishmania. The treatment of cutaneous leishmaniasis is unsatisfactory, thus, much research effort has been focused on investigating new compounds with lower collateral effects to the patients and derived from low-cost sources, such as natural products. In the present study, we evaluated the in vitro directly effect of the flavonoid quercetin against Leishmania (Viannia) braziliensis. Quercetin inhibited the proliferation of promastigote forms at all tested concentrations, these effect were due to increasing the reactive oxygen species (ROS) production, phosphatidylserine exposure and loss of plasma membrane integrity. Moreover, quercetin reduced the number of parasites in L. braziliensis-infected macrophages, reducing the levels of TNF-α and increasing IL-10 synthesis without modulate nitric oxide (NO) production. In addition, quercetin upregulated Nrf2/HO-1 expression and modulated the labile iron pool in infected macrophages, culminating in a depletion of available iron for L. braziliensis replication.

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Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host's blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo.

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BACKGROUND: The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES: Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS: Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS: MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS: Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

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