An RNA-based system to study hepatitis B virus replication and evaluate antivirals.

Hepatitis B virus (HBV) chronically infects an estimated 300 million people, and standard treatments are rarely curative. Infection increases the risk of liver cirrhosis and hepatocellular carcinoma, and consequently, nearly 1 million people die each year from chronic hepatitis B. Tools and approaches that bring insights into HBV biology and facilitate the discovery and evaluation of antiviral drugs are in demand. Here, we describe a method to initiate the replication of HBV, a DNA virus, using synthetic RNA. This approach eliminates contaminating background signals from input virus or plasmid DNA that plagues existing systems and can be used to study multiple stages of HBV replication. We further demonstrate that this method can be uniquely applied to identify sequence variants that confer resistance to antiviral drugs.

Chikungunya Virus and Its Envelope Protein E2 Induce Hyperalgesia in Mice: Inhibition by Anti-E2 Monoclonal Antibodies and by Targeting TRPV1.

Chikungunya virus is an arthropod-borne infectious agent that causes Chikungunya fever disease. About 90% of the infected patients experience intense polyarthralgia, affecting mainly the extremities but also the large joints such as the knees. Chronic disease symptoms persist for months, even after clearance of the virus from the blood. Envelope proteins stimulate the immune response against the Chikungunya virus, becoming an important therapeutic target. We inactivated the Chikungunya virus (iCHIKV) and produced recombinant E2 (rE2) protein and three different types of anti-rE2 monoclonal antibodies. Using these tools, we observed that iCHIKV and rE2 protein induced mechanical hyperalgesia (electronic aesthesiometer test) and thermal hyperalgesia (Hargreaves test) in mice. These behavioral results were accompanied by the activation of dorsal root ganglia (DRG) neurons in mice, as observed by calcium influx. Treatment with three different types of anti-rE2 monoclonal antibodies and absence or blockade (AMG-9810 treatment) of transient receptor potential vanilloid 1 (TRPV1) channel diminished mechanical and thermal hyperalgesia in mice. iCHIKV and rE2 activated TRPV1+ mouse DRG neurons in vitro, demonstrating their ability to activate nociceptor sensory neurons directly. Therefore, our mouse data demonstrate that targeting E2 CHIKV protein with monoclonal antibodies and inhibiting TRPV1 channels are reasonable strategies to control CHIKV pain.

Murine Susceptibility to Leishmania amazonensis Infection Is Influenced by Arginase-1 and Macrophages at the Lesion Site.

Cutaneous leishmaniasis is a zoonotic infectious disease broadly distributed worldwide, causing a range of diseases with clinical outcomes ranging from self-healing infections to chronic disfiguring disease. The effective immune response to this infection is yet to be more comprehensively understood and is fundamental for developing drugs and vaccines. Thus, we used experimental models of susceptibility (BALB/c) and partial resistance (C57BL/6) to Leishmania amazonensis infection to investigate the local profile of mediators involved in the development of cutaneous leishmaniasis. We found worse disease outcome in BALB/c mice than in C57BL/6 mice, with almost 15 times higher parasitic load, ulcerated lesion formation, and higher levels of IL-6 in infected paws. In contrast, C57BL/6 presented higher levels of IFN-γ and superoxide anion (•O2-) after 11 weeks of infection and no lesion ulcerations. A peak of local macrophages appeared after 24 h of infection in both of the studied mice strains, followed by another increase after 240 h, detected only in C57BL/6 mice. Regarding M1 and M2 macrophage phenotype markers [iNOS, MHC-II, CD206, and arginase-1 (Arg-1)], we found a pronounced increase in Arg-1 levels in BALB/c after 11 weeks of infection, whereas C57BL/6 showed an initial predomination of markers from both profiles, followed by an M2 predominance, coinciding with the second peak of macrophage infiltration, 240 h after the infection. Greater deposition of type III collagen and lesion resolution was also observed in C57BL/6 mice. The adoptive transfer of macrophages from C57BL/6 to infected BALB/c at the 11th week showed a reduction in both edema and the number of parasites at the lesion site, in addition to lower levels of Arg-1. Thus, C57BL/6 mice have a more effective response against L. amazonensis, based on a balance between inflammation and tissue repair, while BALB/c mice have an excessive Arg-1 production at late infection. The worst evolution seems to be influenced by recruitment of Arg-1 related macrophages, since the adoptive transfer of macrophages from C57BL/6 mice to BALB/c resulted in better outcomes, with lower levels of Arg-1.

The anti-Zika virus and anti-tumoral activity of the citrus flavanone lipophilic naringenin-based compounds.

Flavonoids are natural products widely recognized for their plurality of applications such as antiviral, antiproliferative, antitumor activities and, antioxidant properties. The flavanone naringenin is presented in citrus fruits and has been studied to combat recurrent diseases that still lack effective treatment. Research groups have been investing efforts to the development of new, safe and active candidates to combat these agents or conditions and despite good results recently reported against the Zika virus (ZIKV) and tumor cells, the use of citrus naringenin is limited due to its low bioavailability. Structural exchanges through functionalization, for example, attaching lipophilic groups instead of hydroxyl groups, can further enhance biological properties. Here, the synthesis and characterization of regioselective naringenin mono-7-O-ethers and both mono and di-fatty acid esters, structurally lipophilic ones were demonstrated. Finally, in vitro studies of anti-ZIKV action and antiproliferative activities against melanoma (B16-F10) and breast carcinoma (4T1) cells showed the ether derivatives were actives, with IC50 ranging from 6.76, 18.5 and 22.6 µM to 28.53, 45.1 and 32.3 µM referring to ZIKV, B16-F10 and 4T1 cell lines, respectively. The lipophilic ethers present the ability to inhibit selectively ZIKV-replication in human cells and inhibitions. This class of modifications in flavonoid molecules could be further explore in the future development of specific anti-ZIKV compounds.

Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus

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.

Genetic and biological characterisation of Zika virus isolates from different Brazilian regions

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.

Macrophage Polarization in Leishmaniasis: Broadening Horizons.

Leishmaniasis is a vector-borne neglected tropical disease that affects more than 700,000 people annually. Leishmania parasites cause the disease, and different species trigger a distinct immune response and clinical manifestations. Macrophages are the final host cells for the proliferation of Leishmania parasites, and these cells are the key to a controlled or exacerbated response that culminates in clinical manifestations. M1 and M2 are the two main macrophage phenotypes. M1 is a pro-inflammatory subtype with microbicidal properties, and M2, or alternatively activated, is an anti-inflammatory/regulatory subtype that is related to inflammation resolution and tissue repair. The present review elucidates the roles of M1 and M2 polarization in leishmaniasis and highlights the role of the salivary components of the vector and the action of the parasite in the macrophage plasticity.

Response to Trypanosoma cruzi by Human Blood Cells Enriched with Dentritic Cells Is Controlled by Cyclooxygenase-2 Pathway.

Chagas disease (Cd) or American human trypanosomiasis is caused by Trypanosoma cruzi and affects ~7 million people, mostly in Latin America. The infective trypomastigote forms of the parasite can invade several human blood cell populations, including monocytes and dendritic cells (DC). Although these cells display a wide functional diversity, their interactions with T. cruzi via cyclooxygenase (COX) and cyclic adenosine monophosphate (cAMP) dependent pathways have not been analyzed. To exploiting this mechanism, DC-enriched peripheral human blood mononuclear cell populations (DC-PBMC) were used as our model. Our results showed that the treatment of these cell populations with celecoxib (CEL), a cyclooxygenase-2 selective inhibitor or SQ 22,536, an adenilate cyclase inhibitor, significantly caused marked inhibition of T. cruzi infection. In contrast, aspirin (ASA, a non-selective COX-1 and COX-2 inhibitor) treatment did not inhibit the infection of the cells by the parasite and was independent of nitric oxide (NO) production. The expression of co-stimulatory molecules CD80 and CD86 were similar on cells treated or not with both COX-inhibitors. The infection stimulated the release of TNF-α, IL-1ß, IL-6, IL-8, and IL-10 production by infected cells. Treatment with ASA or CEL did not affect TNF-α, IL-6, IL-8, IL-10, and NO production by infected cells, but increased IL-1ß production by them. Our results suggest a key role of COX-2 and cAMP pathways in T. cruzi invasion process of human blood cells and these pathways may represent targets of new therapeutic options for Cd.

Trypanosoma cruzi: Inhibition of infection of human monocytes by aspirin.

Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for progression of the parasite life cycle and development of Chagas disease. Prostaglandin E2 (PGE2) and other eicosanoids potently modulate host response and contribute to Chagas disease progression. In this study, we evaluated the effect of aspirin (ASA), a non-selective cyclooxygenase (COX) inhibitor on the T. cruzi invasion and its influence on nitric oxide and cytokine production in human monocytes. The pretreatment of monocytes with ASA or SQ 22536 (adenylate-cyclase inhibitor) induced a marked inhibition of T. cruzi infection. On the other hand, the treatment of monocytes with SQ 22536 after ASA restored the invasiveness of T. cruzi. This reestablishment was associated with a decrease in nitric oxide and PGE2 production, and also an increase of interleukin-10 and interleukin-12 by cells pre-treated with ASA. Altogether, these results reinforce the idea that the cyclooxygenase pathway plays a fundamental role in the process of parasite invasion in an in vitro model of T. cruzi infection.

Activity of rosuvastatin in tachyzoites of Toxoplasma gondii (RH strain) in HeLa cells.

Due to the toxicity of conventional medication in toxoplasmosis, some drugs are being studied for treating this infection, such as statins, especially rosuvastatin compound, which is efficient in inhibiting the initial isoprenoid biosynthesis processes in humans and the parasite. The goal of this study was to assess the activity of rosuvastatin in HeLa cells infected with the RH strain of T. gondii. In the experiment, HeLa cells (1 × 105) were infected with tachyzoites of T. gondii (5 × 105). After the experimental infection, we assessed the number of infected cells and the amount of intracellular tachyzoites. In addition, culture supernatants were collected to determine the amount of cytokines by cytometric bead array. We observed that there was no cytotoxicity in the concentrations tested in this cell line. The effect of rosuvastatin showed a significant reduction in both the number of infected cells and the proliferation index of the intracellular parasite, when compared with the conventional treatment combining sulfadiazine and pyrimethamine for toxoplasmosis. There were also reduced levels of cytokines IL-6 and IL-17. Therefore, it was concluded that rosuvastatin exhibited antiproliferative activity. The data presented are significant to promote further studies and the search for alternative treatment for toxoplasmosis.

Genome-wide analyses reveal a highly conserved Dengue virus envelope peptide which is critical for virus viability and antigenic in humans.

Targeting regions of proteins that show a high degree of structural conservation has been proposed as a method of developing immunotherapies and vaccines that may bypass the wide genetic variability of RNA viruses. Despite several attempts, a vaccine that protects evenly against the four circulating Dengue virus (DV) serotypes remains elusive. To find critical conserved amino acids in dengue viruses, 120 complete genomes of each serotype were selected at random and used to calculate conservation scores for nucleotide and amino acid sequences. The identified peptide sequences were analysed for their structural conservation and localisation using crystallographic data. The longest, surface exposed, highly conserved peptide of Envelope protein was found to correspond to amino acid residues 250 to 270. Mutation of this peptide in DV1 was lethal, since no replication of the mutant virus was detected in human cells. Antibodies against this peptide were detected in DV naturally infected patients indicating its potential antigenicity. Hence, this study has identified a highly conserved, critical peptide in DV that is a target of antibodies in infected humans.

The citrus flavonone naringenin reduces lipopolysaccharide-induced inflammatory pain and leukocyte recruitment by inhibiting NF-κB activation.

Lipopolysaccharide (LPS) is the major structural component of Gram-negative bacteria cell wall and a highly pro-inflammatory toxin. Naringenin is found in Citrus fruits and exhibits antioxidant and anti-inflammatory properties through inhibition of NF-κB activation but its effects in LPS-induced inflammatory pain and leukocyte recruitment were not investigated yet. We investigated the effects of naringenin in mechanical hyperalgesia, thermal hyperalgesia and leukocyte recruitment induced by intraplantar injection of LPS in mice. We found that naringenin reduced hyperalgesia to mechanical and thermal stimuli, myeloperoxidase (MPO, a neutrophil and macrophage marker) and N-acetyl-ß-D-glucosaminidase (NAG, a macrophage marker) activities, oxidative stress and cytokine (TNF-α, IL-1ß, IL-6, and IL-12) production in the paw skin. In the peritoneal cavity, naringenin reduced neutrophil and mononuclear cell recruitment, and abrogated MPO and NAG activity, cytokine and superoxide anion production, and lipid peroxidation. In vitro, pre-treatment with naringenin inhibited superoxide anion and cytokine (TNF-α, IL-1ß, IL-6, and IL-12) production by LPS-stimulated RAW 264.7 macrophages. Finally, we demonstrated that naringenin inhibited NF-κB activation in vitro and in vivo. Therefore, naringenin is a promising compound to treat LPS-induced inflammatory pain and leukocyte recruitment.

Quinic acid derivatives inhibit dengue virus replication in vitro.

BACKGROUND: Dengue is the most prevalent arboviral disease in tropical and sub-tropical areas of the world. The incidence of infection is estimated to be 390 million cases and 25,000 deaths per year. Despite these numbers, neither a specific treatment nor a preventive vaccine is available to protect people living in areas of high risk. RESULTS: With the aim of seeking a treatment that can mitigate dengue infection, we demonstrated that the quinic acid derivatives known as compound 2 and compound 10 were effective against all four dengue virus serotypes and safe for use in a human hepatoma cell line (Huh7.5). Both compounds were non-virucidal to dengue virus particles and did not interfere with early steps of the dengue virus life cycle, including binding and internalization. Experiments using a replicon system demonstrated that compounds 2 and 10 impaired dengue virus replication in Huh7.5 cells. Additionally, the anti-dengue virus effects of the quinic acid derivatives were preserved in human peripheral blood mononuclear cells. CONCLUSIONS: Taken together, these data suggest that quinic acid derivatives represent a novel chemical class of active compounds that could be used to combat dengue virus infection.

Immature dendritic cells generated from cryopreserved human monocytes show impaired ability to respond to LPS and to induce allogeneic lymphocyte proliferation.

Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs), the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp)-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh)-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h) and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.

Dendritic cell apoptosis and the pathogenesis of dengue.

Dengue viruses and other members of the Flaviviridae family are emerging human pathogens. Dengue is transmitted to humans by Aedes aegypti female mosquitoes. Following infection through the bite, cells of the hematopoietic lineage, like dendritic cells, are the first targets of dengue virus infection. Dendritic cells (DCs) are key antigen presenting cells, sensing pathogens, processing and presenting the antigens to T lymphocytes, and triggering an adaptive immune response. Infection of DCs by dengue virus may induce apoptosis, impairing their ability to present antigens to T cells, and thereby contributing to dengue pathogenesis. This review focuses on general mechanisms by which dengue virus triggers apoptosis, and possible influence of DC-apoptosis on dengue disease severity.

Screening of Dengue virus antiviral activity of marine seaweeds by an in situ enzyme-linked immunosorbent assay.

Dengue is a significant public health problem worldwide. Despite the important social and clinical impact, there is no vaccine or specific antiviral therapy for prevention and treatment of dengue virus (DENV) infection. Considering the above, drug discovery research for dengue is of utmost importance; in addition natural marine products provide diverse and novel chemical structures with potent biological activities that must be evaluated. In this study we propose a target-free approach for dengue drug discovery based on a novel, rapid, and economic in situ enzyme-linked immunosorbent assay and the screening of a panel of marine seaweed extracts. The in situ ELISA was standardized and validated for Huh7.5 cell line infected with all four serotypes of DENV, among them clinical isolates and a laboratory strain. Statistical analysis showed an average S/B of 7.2 and Z-factor of 0.62, demonstrating assay consistency and reliability. A panel of fifteen seaweed extracts was then screened at the maximum non-toxic dose previously determined by the MTT and Neutral Red cytotoxic assays. Eight seaweed extracts were able to reduce DENV infection of at least one serotype tested. Four extracts (Phaeophyta: Canistrocarpus cervicornis, Padina gymnospora; Rhodophyta: Palisada perforate; Chlorophyta: Caulerpa racemosa) were chosen for further evaluation, and time of addition studies point that they might act at an early stage of the viral infection cycle, such as binding or internalization.

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

Brevidensoviruses have an encapsidated, single-stranded DNA genome that predominantly has a negative polarity. In recent years, they have received particular attention due to their potential role in the biological control of pathogenic arboviruses and to their unnoticed presence in cell cultures as contaminants. In addition, brevidensoviruses may also be useful as viral vectors. This study describes the first genetic and biological characterization of a mosquito densovirus that was isolated in Brazil; moreover, we examined the phylogenetic relationship between this isolate and the other brevidensoviruses. We further demonstrate that this densovirus has the potential to be used to biologically control dengue virus (DENV) infection with in vitro co-infection experiments. The present study provides evidence that this densovirus isolate is a fast-spreading virus that affects cell growth and DENV infection.

Dengue virus type 3 isolated from a fatal case with visceral complications induces enhanced proinflammatory responses and apoptosis of human dendritic cells.

A recent (2007 to 2009) dengue outbreak caused by dengue virus (DENV) in Paraguay presented unusual severe clinical outcomes associated with 50% mortality rates. Although it has been reported that inflammatory responses influence the severity of dengue virus infection (T. Pang, M. J. Cardosa, and M. G. Guzman, Immunol. Cell Biol. 85:43-45, 2007), there remains a paucity of information on virus-innate immunity interactions influencing clinical outcome. Using human dendritic cells from a major innate immune cell population as an in vitro model, we have investigated signature cytokine responses as well as infectivity-replicative profiles of DENV clinical isolates from either a nonfatal case of classical dengue fever (strain DENV3/290; isolated in Brazil in 2002) or a fatal case of dengue fever with visceral complications isolated in Paraguay in 2007 (strain DENV3/5532). Strain DENV3/5532 was found to display significantly higher replicative ability than DENV3/290 in monocyte-derived dendritic cells (mdDCs). In addition, compared to DENV3/290 results, mdDCs exposed to DENV3/5532 showed increased production of proinflammatory cytokines associated with higher rates of programmed cell death, as shown by annexin V staining. The observed phenotype was due to viral replication, and tumor necrosis factor alpha (TNF-α) appears to exert a protective effect on virus-induced mdDC apoptosis. These results suggest that the DENV3/5532 strain isolated from the fatal case replicates within human dendritic cells, modulating cell survival and synthesis of inflammatory mediators.

Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.