Low CCL2 and CXCL8 Production and High Prevalence of Allergies in Children with Microcephaly Due to Congenital Zika Syndrome.

Congenital Zika Syndrome (CZS) is associated with an increased risk of microcephaly in affected children. This study investigated the peripheral dysregulation of immune mediators in children with microcephaly due to CZS. Gene expression quantified by qPCR in whole blood samples showed an increase in IFNγ and IL-13 transcripts in children affected with microcephaly compared to the control group. The microcephaly group exhibited significantly decreased CCL2 and CXCL8 levels in serum, quantified by CBA assay. An allergic profile questionnaire revealed a high prevalence of allergies in the microcephaly group. In accordance, elevated serum IgE level measured by the Proquantum Immunoassay was observed in children affected with microcephaly compared to the control group. Altogether, these findings show a persistent systemic inflammation in children with microcephaly due to CZS and suggest a possible impairment in leukocyte migration caused by low production of CCL2 and CXCL8, in addition to high levels of IgE associated with high prevalence of allergies. The dysregulation of inflammatory genes and chemokines underscores the importance of understanding the immunological characteristics of CZS. Further investigation into the long-term consequences of systemic inflammation in these children is crucial for developing appropriate therapeutic strategies and tailored vaccination protocols.

Biocomposite for Prolonged Release of Water-Soluble Drugs.

This study aimed to develop a prolonged-release system based on palygorskite and chitosan, which are natural ingredients widely available, affordable, and accessible. The chosen model drug was ethambutol (ETB), a tuberculostatic drug with high aqueous solubility and hygroscopicity, which is incompatible with other drugs used in tuberculosis therapy. The composites loaded with ETB were obtained using different proportions of palygorskite and chitosan through the spray drying technique. The main physicochemical properties of the microparticles were determined using XRD, FTIR, thermal analysis, and SEM. Additionally, the release profile and biocompatibility of the microparticles were evaluated. As a result, the chitosan-palygorskite composites loaded with the model drug appeared as spherical microparticles. The drug underwent amorphization within the microparticles, with an encapsulation efficiency greater than 84%. Furthermore, the microparticles exhibited prolonged release, particularly after the addition of palygorskite. They demonstrated biocompatibility in an in vitro model, and their release profile was influenced by the proportion of inputs in the formulation. Therefore, incorporating ETB into this system offers improved stability for the administered product in the initial tuberculosis pharmacotherapy dose, minimizing its contact with other tuberculostatic agents in the treatment, as well as reducing its hygroscopicity.

Nanocomposite gels of poloxamine and Laponite for ß-Lapachone release in anticancer therapy.

Nano-hybrid systems have been shown to be an attractive platform for drug delivery. Laponite® RD (LAP), a biocompatible synthetic clay, has been exploited for its ability to establish of strong secondary interactions with guest compounds and hybridization with polymers or small molecules that improves, for instance, cell adhesion, proliferation, and differentiation or facilitates drug attachment to their surfaces through charge interaction. In this work, LAP was combined with Tetronics, X-shaped amphiphilic PPO-PEO (poly (propylene oxide)-poly (ethylene oxide) block copolymers. ß-Lapachone (BLPC) was selected for its anticancer activity and its limited bioavailability due to very low aqueous solubility, with the aim to improve this by using LAP/Tetronic nano-hybrid systems. The nanocarriers were prepared over a range of Tetronic 1304 concentrations (1 to 20% w/w) and LAP (0 to 3% w/w). A combination of physicochemical methods was employed to characterize the hybrid systems, including rheology, particle size and shape (DLS, TEM), thermal analysis (TG and DSC), FTIR, solubility studies and drug release experiments. In vitro cytotoxicity assays were performed with BALB/3T3 and MCF-7 cell lines. In hybrid systems, a sol-gel transition can occur below physiological temperature. BLPC exhibits the most significant increase in solubility in formulations with a high concentration of T1304 (over 10% w/w) and 1.5% w/w LAP, or systems with only LAP (1.5%), with a 50 and 100-fold increase in solubilisation, respectively. TEM images showed spherical micelles of T1304, which elongated into wormlike micelles with concentration (20%) and in the presence of LAP, a finding that has not been reported before. A sustained release of BLPC over 140 hours was achieved in one of the formulations (10% T1304 with 1.5% laponite), which also showed the best selectivity index towards cancer cells (MCF-7) over BALB/3T3 cell lines. In conclusion, BLPC-loaded T1304/LAP nano-hybrid systems proved safe and highly effective and are thus a promising formulation for anticancer therapy.

Chloroquine as a promising adjuvant therapy for type 1 Diabetes Mellitus.

Chloroquine (CQ) and hydroxychloroquine, are promising anti-inflammatory drugs for the treatment of Diabetes mellitus (DM) to prevent associated complications. Therefore, this study evaluated the anti-inflammatory effects of CQ-free and CQ-incorporated polylactic acid nanoparticles (NPs) in the peripheral blood mononuclear cells (PBMCs) of patients with type 1 Diabetes mellitus (T1DM). In total, 25 normoglycemic individuals and 25 patients with T1DM aged 10-16 years were selected and glycemic controls evaluated. After cell viability assessed by MTT assay, T1DM PBMCs were subjected to a CQ concentration of 10 µM in three different conditions: not treated (NT), treated with CQ, and treated with CQ NPs. The cells were incubated for 48 h, and the mRNA expressions of cytokines IL1B, IFNG, TNFA, IL12, and IL10 were determined by relative quantification through real-time PCR at 24 h intervals. IL1B expression decreased in CQ and CQ NP-treated cells after 48 h (p < 0.001) and 24 h (p < 0.05) of treatment, respectively. IFNG and IL12 expressions significantly decreased (p < 0.001) in cells treated with CQ and CQ NPs at 24 and 48 h compared to NT. TNFA and IL10 expressions significantly decreased after 48 h (p < 0.001) and 24 h (p < 0.002), respectively, by both CQ and CQ NPs treatment. Despite being a preliminary in vitro study, CQ has anti-inflammatory activity in the primary cells of T1DM patients and could represent an alternative and adjuvant anti-inflammatory therapy to prevent diabetes complications.

Tailoring structural properties of spray-dried methotrexate-loaded poly (lactic acid)/poloxamer microparticle blends.

Drug delivery systems can overcome cancer drug resistance, improving the efficacy of chemotherapy agents. Poly (lactic acid) (PLA) microparticles are an interesting alternative because their hydrophobic surface and small particle size could facilitate interactions with cells. In this study, two poloxamers (PLX 407 and 188) were applied to modulate the structural features, the drug release behavior and the cell viability from spray-dried microparticles. Five formulations with different PLA: PLX blend ratio (100:0, 75:25, 50:50, 25:50, and 0:100) were well-characterized by SEM, particle size analysis, FTIR spectroscopy, differential scanning calorimetry (DSC), and X-ray diffraction analysis (XRD). The spray-dried microparticles showed higher drug loading, spherical-shape, and smaller particle size. The type of poloxamer and blend ratio affected their structural and functional properties such as morphology, crystallinity, blend miscibility, drug release rate, and cell viability. The methotrexate (MTX), a model drug, was loaded in amorphous spray-dried microparticles. Moreover, the drug release studies demonstrated that PLX induced a leaching-effect of MTX from PLA: PLX blends, suggesting the formation of MTX/PLX micelles in aqueous medium. This finding was better established by cell viability assays. Therefore, biocompatible PLA: PLX blends showed promising in vitro results, and further in vivo studies will be performed to evaluate the performance of this chemotherapeutic agent.

Brefeldin A and Cytochalasin B reduce dengue virus replication in cell cultures but do not protect mice against viral challenge.

BACKGROUND: Dengue is an emerging arboviral disease caused by dengue virus (DENV). DENV belongs to the family Flaviviridae and genus Flavivirus. No specific anti-DENV drugs are currently available. METHODS: We investigated the antiviral activity of Brefeldin A (BFA) and Cytochalasin B (CB) against this infection. The drugs BFA and CB were used in the in vitro treatment of dengue-2 virus (DENV-2) infections in Vero cell cultures and in protection from lethality by post-challenge administration in Swiss mice. Viral load was quantified by qRT-PCR and plaque assay in Vero cell cultures, post-infection, treated or not with the drugs. Post-challenge drug levels were evaluated by survival analysis. RESULTS: Our results indicate that doses of 5 µg ml-1 of BFA and 10 µg ml-1 of CB are not toxic to the cells and induce a statistically significant inhibition of DENV-2 replication in Vero cells when compared to control. No BFA- or CB-treated mice survived the challenge with DENV-2. CONCLUSION: These data suggest that BFA and CB have an antiviral action against DENV-2 replication in Vero cell culture, but do not alter infected mice mortality.

Improving Encapsulation of Hydrophilic Chloroquine Diphosphate into Biodegradable Nanoparticles: A Promising Approach against Herpes Virus Simplex-1 Infection.

Chloroquine diphosphate (CQ) is a hydrophilic drug with low entrapment efficiency in hydrophobic nanoparticles (NP). Herpes simplex virus type 1 (HSV-1) is an enveloped double-stranded DNA virus worldwide known as a common human pathogen. This study aims to develop chloroquine-loaded poly(lactic acid) (PLA) nanoparticles (CQ-NP) to improve the chloroquine anti- HSV-1 efficacy. CQ-NP were successfully prepared using a modified emulsification-solvent evaporation method. Physicochemical properties of the NP were monitored using dynamic light scattering, atomic force microscopy, drug loading efficiency, and drug release studies. Spherical nanoparticles were produced with modal diameter of <300 nm, zeta potential of -20 mv and encapsulation efficiency of 64.1%. In vitro assays of CQ-NP performed in Vero E6 cells, using the MTT-assay, revealed different cytotoxicity levels. Blank nanoparticles (B-NP) were biocompatible. Finally, the antiviral activity tested by the plaque reduction assay revealed greater efficacy for CQ-NP compared to CQ at concentrations equal to or lower than 20 µg mL-1 (p < 0.001). On the other hand, the B-NP had no antiviral activity. The CQ-NP has shown feasible properties and great potential to improve the antiviral activity of drugs.

Immune Response to Human Papillomavirus One Year after Prophylactic Vaccination with AS04-Adjuvanted HPV-16/18 Vaccine: HPV-Specific IgG and IgA Antibodies in the Circulation and the Cervix

Objective: This study was designed to describe the course of IgG/IgA responses in cervical secretions and in serum one year after the first dose of intramuscular administration of the HPV16/18 AS04-adjuvant vaccine. Methods: Blood and cervical mucus samples were collected for immunologic assays, 7 months after the first doses and 1 year following the last boost vaccination (month 7) by enzyme linked immunosorbent assay (ELISA). The detection of IgG and IgA anti-HPV/VLP was developed for this purpose. Result: A total of 100% of serum samples were IgG antibody positive at a titer of 1:100 at both time periods and decreased according to the serum dilution. For serum IgA antibody, 95% were positive one month after vaccination and 79% were positive 1 year later. Similar results were observed with the cervical samples positive for both IgG and IgA antibodies at one month and decreasing after 1 year to 33% and 29%. The median absorbance in serum and the cervix for IgG and IgA anti-HPV-VLP antibodies was significantly higher at one month after vaccination when compared to 1 year post-vaccination (P<0.0001). Conclusion: Immune responses were significant one year after immunization, however it decreased in cervical and serum samples when compared to levels observed one month after the last dose. This suggests that a vaccine booster may be necessary to increase antibody titers.

Human Herpesvirus 8 in Perinatally HIV-infected Children with Interstitial Lung Disease.

INTRODUCTION: Human herpesvirus 8 (HHV-8) is associated with the pathogenesis of Kaposi Sarcoma and interstitial pneumonitis in adults. This study aims to evaluate association between HHV-8 and interstitial lung disease in HIV-infected children. METHODS: HIV-infected children with interstitial pneumonitis underwent lung biopsies in a tertiary hospital and were investigated for HHV-8, Epstein-Barr virus (EBV) and cytomegalovirus (CMV) using polymerase chain reaction (PCR) and immunohistochemistry in lung tissue. Peripheral blood PCR was also performed for HHV-8. RESULTS: From six patients included, PCR for HHV-8 was positive in lung samples in four children and in peripheral blood in one. PCR for EBV and CMV and immunohistochemical study for HHV-8, EBV and CMV in lung were negative in all patients. CONCLUSION: No previous cases of HHV-8-associated interstitial pneumonitis was described in HIV-infected children. An immunological disorder and an infectious agent might influence development of the lymphoid interstitial pneumonitis. HHV-8 may be this infectious trigger.

Human herpesvirus 8 (HHV-8) detected by nested polymerase chain reaction (PCR) in HIV patients with or without Kaposi's sarcoma. An analytic cross-sectional study / Herpesvírus humano 8 (HHV-8) detectado por reação da polimerase em cadeia do tipo nested PCR em pacientes HIV-positivos com ou sem sarcoma de Kaposi. Um estudo transversal analítico

CONTEXT AND OBJECTIVE: Kaposi's sarcoma (KS) is a common neoplastic disease in AIDS patients. The aim of this study was to evaluate the frequency of human herpesvirus 8 (HHV-8) infection in human immunodeficiency virus (HIV)-infected patients, with or without KS manifestations and correlate HHV-8 detection with KS staging. DESIGN AND SETTING: Analytic cross-sectional study conducted in a public tertiary-level university hospital in Ribeirão Preto, São Paulo, Brazil. METHODS: Antibodies against HHV-8 lytic-phase antigens were detected by means of the immunofluorescence assay. HHV-8 DNA was detected in the patient samples through a nested polymerase chain reaction (nested PCR) that amplified a region of open reading frame (ORF)-26 of HHV-8. RESULTS: Anti-HHV-8 antibodies were detected in 30% of non-KS patients and 100% of patients with KS. Furthermore, the HHV-8 DNA detection rates observed in HIV-positive patients with KS were 42.8% in serum, 95.4% in blood samples and 100% in skin biopsies; and in patients without KS, the detection rate was 4% in serum. Out of the 16 serum samples from patients with KS-AIDS who were classified as stage II, two were positive (12.5%); and out of the 33 samples from patients in stage IV, 19 (57.6%) were positive. CONCLUSION: We observed an association between HHV-8 detection and disease staging, which was higher in the serum of patients in stage IV. This suggests that detection of HHV-8 DNA in serum could be very useful for clinical assessment of patients with KS and for monitoring disease progression.

CONTEXTO E OBJETIVO: Sarcoma de Kaposi (SK) é uma doença neoplásica comum em pacientes com aids. O objetivo deste estudo foi avaliar a frequência da infecção por herpesvírus humano 8 (HHV-8) em pacientes infectados por HIV, com ou sem SK e associar a detecção do HHV-8 com o estadiamento do SK. TIPO DE ESTUDO E LOCAL: Estudo transversal analítico realizado em hospital universitário público terciário de Ribeirão Preto, São Paulo, Brasil. MÉTODOS: Anticorpos contra antígenos de fase lítica do HHV-8 foram detectados por imunofluorescência. O DNA viral de HHV-8 foi detectado em amostras de pacientes pela reação em cadeia da polimerase do tipo nested (nested PCR), que amplificou uma região do fragmento de leitura aberta (ORF)-26 do HHV-8. RESULTADOS: Anticorpos anti-HHV-8 foram detectados em 30% dos pacientes sem SK e 100% dos com SK. Além disso, a detecção de HHV-8 DNA observada em pacientes HIV-positivos com SK foi de 42,8% no soro, 95,4% em amostras de sangue e 100% em biópsias de pele, e em pacientes sem SK foi de 4% no soro. Das 16 amostras de soro de pacientes com SK-AIDS classificados como estádio II, duas foram positivas (12,5%) e, das 33 amostras de pacientes no estádio IV, 19 (57,6%) foram positivas. CONCLUSÃO: Observamos associação entre a detecção do HHV-8 e o estadiamento da doença, que foi maior no soro de pacientes no estágio IV. Isso sugere que a detecção do HHV-8 no soro poderia ser muito útil para a avaliação clínica de pacientes com SK e para o monitoramento da progressão da doença.

Human Papillomavirus Vaccine-Induced Cytokine Messenger RNA Expression in Vaccinated Women.

The objective of this work was to evaluate the influence of human papillomavirus (HPV) vaccination on peripheral blood mononuclear cell (PBMC) proliferation and cytokine gene transcription. PBMCs isolated after HPV immunization were incubated with HPV vaccine, phytohemagglutinin, or buffer. Cell proliferation was assessed by MTT reduction assay. RNA was extracted from PBMCs, and the relative concentration of cytokine messenger RNA (mRNA) transcripts (IFN-ß, IFN-γ, IL-12, TNF-α, IL-6, IL-17, or IL-10) relative to transcription of the ß-actin gene was determined by real-time polymerase chain reaction. PBMC proliferation in response to HPV vaccine and PHA were greater than that observed in unstimulated cells (p<0.001). Cytokine mRNAs were upregulated in stimulated PBMC cultures. The median increase in vaccine-stimulated cultures was: IFN-ß=334.4-fold; IL-12=46.33-fold; IFN-γ=12.64-fold; IL-6=9.07-fold; IL-17=7.33-fold; IL-10=6.47-fold; and TNF-α=2.36-fold. The IFN-ß expression was significantly higher (p<0.05). Proliferative PBMC responses and multiple cytokine gene expression were detected in women who received the HPV vaccine.

Human herpesvirus 8 (HHV-8) detected by nested polymerase chain reaction (PCR) in HIV patients with or without Kaposi's sarcoma. An analytic cross-sectional study.

CONTEXT AND OBJECTIVE: Kaposi's sarcoma (KS) is a common neoplastic disease in AIDS patients. The aim of this study was to evaluate the frequency of human herpesvirus 8 (HHV-8) infection in human immunodeficiency virus (HIV)-infected patients, with or without KS manifestations and correlate HHV-8 detection with KS staging. DESIGN AND SETTING: Analytic cross-sectional study conducted in a public tertiary-level university hospital in Ribeirão Preto, São Paulo, Brazil. METHODS: Antibodies against HHV-8 lytic-phase antigens were detected by means of the immunofluorescence assay. HHV-8 DNA was detected in the patient samples through a nested polymerase chain reaction (nested PCR) that amplified a region of open reading frame (ORF)-26 of HHV-8. RESULTS: Anti-HHV-8 antibodies were detected in 30% of non-KS patients and 100% of patients with KS. Furthermore, the HHV-8 DNA detection rates observed in HIV-positive patients with KS were 42.8% in serum, 95.4% in blood samples and 100% in skin biopsies; and in patients without KS, the detection rate was 4% in serum. Out of the 16 serum samples from patients with KS-AIDS who were classified as stage II, two were positive (12.5%); and out of the 33 samples from patients in stage IV, 19 (57.6%) were positive. CONCLUSION: We observed an association between HHV-8 detection and disease staging, which was higher in the serum of patients in stage IV. This suggests that detection of HHV-8 DNA in serum could be very useful for clinical assessment of patients with KS and for monitoring disease progression.

Structural characterization of a novel peptide with antimicrobial activity from the venom gland of the scorpion Tityus stigmurus: Stigmurin.

A new antimicrobial peptide, herein named Stigmurin, was selected based on a transcriptomic analysis of the Brazilian yellow scorpion Tityus stigmurus venom gland, an underexplored source for toxic peptides with possible biotechnological applications. Stigmurin was investigated in silico, by circular dichroism (CD) spectroscopy, and in vitro. The CD spectra suggested that this peptide interacts with membranes, changing its conformation in the presence of an amphipathic environment, with predominance of random coil and beta-sheet structures. Stigmurin exhibited antibacterial and antifungal activity, with minimal inhibitory concentrations ranging from 8.7 to 69.5µM. It was also showed that Stigmurin is toxic against SiHa and Vero E6 cell lines. The results suggest that Stigmurin can be considered a potential anti-infective drug.

Antiviral activity of chloroquine against dengue virus type 2 replication in Aotus monkeys.

Dengue virus (DENV) of the Flaviviridae family is a single positive-stranded RNA virus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes. The objective of this study was to investigate the use of chloroquine (CLQ) as an antiviral drug against dengue virus in monkeys. To analyze the action of the drug in vivo, nonhuman primates groups (Aotus azarai infulatus) were inoculated with a subcutaneous injection of a virulent strain of DENV-2, treated and untreated CLQ. Blood hematological, viremia, and serum biochemical values were obtained from 16 DENV-2-inoculated, treated and untreated; four received only CLQ and one mock-infected Aotus monkeys. Monkey serum samples (day 0-10 post-inoculation) were assayed by reverse transcription polymerase chain reaction and Cytometric Bead Array for determination of viremia and inflammatory cytokines, respectively. Additionally, body temperature and activity levels were determined. In the present work, CLQ was effective on replication of DENV-2 in Aotus monkeys; a time viremia reduction was observed compared with the controls. The concentration of tumor necrosis factor alpha and interferon gamma in the serum of the animals had a statistically significant reduction in the groups treated with CLQ after infection compared with the controls. A significant decrease in systemic levels of the liver enzyme aspartate aminotransferase (AST) was also observed in the animals treated with CLQ after infection compared with the controls. These results suggest that CLQ interferes in DENV-2 replication in Aotus monkeys.

Detection of immunoglobulin IgA and IgG against human papilloma virus.

The interest in human papilloma virus (HPV) seropositivity has increased considerably since HPV vaccines have become available worldwide. The aim of this study was to assess the performance of enzyme-linked immunosorbent assay (ELISA) in analyzing serum samples provided from women with and without genital DNA-HPV infection confirmed by polymerase chain reaction (PCR), for detection of specific antibodies of the isotypes IgG and IgA recognizing HPV-16 and -18, as well as virus-like particles (VLPs). From August to December 2013, 50 sexually active female patients between 18 and 35 years of age from the outpatient clinic at the university hospital were enrolled. In order to test them, positive controls were obtained from patients with HPV-induced lesions and who were DNA-HPV positive confirmed by PCR. A specific assay was used to identify antibodies to HPV VLPs by ELISA. The samples were divided into HPV positive and negative, and an ELISA detecting IgA and IgG anti-HPV-VLP was carried out. The effectiveness of ELISA and the kappa (k) index was obtained from the values entered in the receiver operating characteristic (ROC) curves for IgG and IgA. IgG-VLP-HPV-16 showed a good correlation between ELISA and PCR (k=0.75), and IgG-VLP-HPV-18 showed a very good correlation between ELISA and PCR (k=0.84). While the IgA antibody correlation was also positive, although weaker, IgA-VLP-HPV-16 was moderate (k=0.45) and IgA-VLP-HPV-18 good (k=0.66). The efficacy of the assay concerning IgG was: sensitivity, specificity, and accuracy were 82.3%, 92%, and 88% to IgG-VLP-HPV-16, and 100%, 92%, and 94% to IgG-VLP-HPV-18. The assay concerning IgA was: sensitivity, specificity, and accuracy were 64.7%, 80%, and 73.8% to IgA-VLP-HPV-16, and 100%, 80%, and 84.8% to IgA-VLP-HPV-18. IgG and IgA antibodies against HPV-16 and -18 can be detected in unvaccinated individuals by using the VLP that serve as the basis for bivalent HPV vaccine. The values for ELISA assays and the values found for IgG correlate good/very good with HPV-16/18 detected by PCR.

Disseminated Kaposi's sarcoma in patients with HIV infection correlates to high serum levels of IL-10.

Human herpesvirus 8 (HHV-8) is the etiologic agent of all Kaposi's sarcoma (KS), the outcome of which is associated with immuno-dysregulation, resulting in the abnormal production of inflammatory cytokines and chemokines. We quantified by enzyme-linked immunosorbent assay serum levels of interleukin (IL)-10, IL-17, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α from patients with KS-AIDS, classic KS, and human immunodeficiency virus (HIV) without KS. A correlation between HHV-8 molecular detection and cytokine production was also performed. We observed that IL-10 production was higher in patients with KS-AIDS when compared to those with classic KS or HIV. However, no significant differences were seen for IFN-γ, TNF-α, or IL-17 production between studied groups. When patients with KS-AIDS were analyzed according to lesion topography, IL-10 levels were higher in patients with disseminated disease than those observed in patients with only cutaneous lesions or cutaneous and digestive and/or respiratory tract lesions. Finally, patients with KS-AIDS that presented viral DNA for HHV-8 in serum showed a higher production of IL-10 when compared with those patients with a negative result for nested polymerase chain reaction for the virus. The results presented here are the first to demonstrate that there exists a stratification of patients with KS-AIDS according to lesion topography where IL-10 levels are higher in those individuals with disseminated disease than those with only localized lesions.

Chloroquine interferes with dengue-2 virus replication in U937 cells.

The objective of this study was to investigate the use of chloroquine (CLQ) as an antiviral agent against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of U937 cells infected with dengue virus type 2 (DENV-2). Viral replication was assessed by quantification of virus produced through detection of copy numbers of DENV-2 RNA, plaque assay and indirect immunofluorescence. qRT-PCR and plaque assays were used to quantify the DENV-2 load in infected U937 cells after CLQ treatment. It was found that a dose of 50 µg/mL of CLQ was not toxic to the cells and resulted in significantly less virus production in infected U937 cells than occurred in untreated cells. In the present work, CLQ was effective against DENV-2 replication in U937 cells, and also caused a statistically significant reduction in expression of proinflammatory cytokines. The present study indicates that CLQ may be used to reduce viral yield in U937 cells.

Chloroquine inhibits dengue virus type 2 replication in Vero cells but not in C6/36 cells.

Dengue viruses are the most important arthropod-borne viruses in terms of morbidity and mortality in the world. Since there is no dengue vaccine available for human use, we have set out to investigate the use of chloroquine as an antiviral drug against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of Vero and C6/36 cells infected with dengue virus type 2 (DENV-2). Real-time RT-PCR and plaque assays were used to quantify the DENV-2 load in infected Vero and C6/36 cells after chloroquine treatment. Our results showed that a dose of 50 µg/ml of chloroquine was not toxic to the cells and induced a statistically significant inhibition of virus production in infected Vero cells when compared to untreated cells. In C6/36 cells, chloroquine does not induce a statistically significant difference in viral replication when compared to untreated cells, showing that this virus uses an unlikely pathway of penetration in these cells, and results were also confirmed by the plaque assay (PFU). These data suggest that the inhibition of virus infection induced by chloroquine is due to interference with acidic vesicles in mammalian cells.

High diversity of HHV-8 molecular subtypes in the Amazon region of Brazil: evidence of an ancient human infection.

The present study describes the molecular epidemiology of Human herpesvirus 8 (HHV-8) among four Indian tribes (Kararao, Arara Laranjal, Tiriyo, and Zo'e) of the Amazon region of Brazil and a group of HIV-1-infected subjects from the urban population of Belem, Para. Infection was characterized by the presence of antibodies using ELISA (measuring antibodies to ORF59, ORF65, K8.1A, K8.1B, and ORF73), and molecular assays (gene amplification of the regions ORF26 and the variable region VR1). Antibodies to HHV-8 were detected in 66 samples of the 221 Brazilian Amerindians, namely, 6 (25%) in the Kararao, 18 (19.6%) in the Arara Laranjal, 24 (42.9%) in the Tiriyo, and 18 (36.7%) in the Zo'e. Among the 477 HIV-1-infected subjects, antibodies to HHV-8 were present in 74 (15.5%) persons. The ORF26 region was amplified in seven samples, one of the Arara Laranjal, one of the Tiriyo, two of the Zo'e, and three of the HIV-1-infected group. Subtyping of HHV-8 described a high multiplicity of molecular subtypes, including C (Zo'e), E (Tiriyo), and B (HIV-1 infected). Serological results confirm the high prevalence of HHV-8 among Amerindians and the presence of three subtypes in the Amazon region of Brazil, including a unique subtype, which favors the idea of HHV-8 as an ancient human infection within this particular geographical region.