C1q modulation of antibody-dependent enhancement of dengue virus infection in human myeloid cell lines is dependent on cell type and antibody specificity.

Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is one of the mechanisms contributing to increased severity during heterotypic, secondary infection. The complement protein C1q has been shown to reduce the magnitude of ADE in vitro. Therefore, we investigated the mechanisms of C1q modulation of ADE, focusing on processes of viral entry. Using a model of ADE of DENV-1 infection in human myeloid cell lines in the presence of monoclonal antibodies, 4G2 and 2H2, we found that C1q produced nearly a 40-fold reduction of ADE of DENV-1 in K562 cells, but had no effect in U937 cells. In K562 cells, C1q reduced adsorption of DENV-1/4G2 and exerted a dual inhibitory effect on adsorption and internalization of DENV-1/2H2. Distinct endocytic pathways in the presence of antibody corresponded to conditions where C1q produced a differential action. Also, C1q did not affect the intrinsic cell response mediated by FcγR in human myeloid cells. The modulation of ADE of DENV-1 by C1q is dependent on the FcγR expressed on immune cells and the specificity of the antibody comprising the immune complex. Understanding protective and pathogenic mechanisms in the humoral response to DENV infections is crucial for the successful design of antivirals and vaccines.

Epidemiological and clinical features of the 2016-2018 Zika virus outbreak in northern Argentina.

BACKGROUND: During the American epidemic, Zika virus (ZIKV) expanded rapidly through dengue virus (DENV)-endemic regions. We analyzed the presentation of ZIKV infection in patients from the City of Orán, Argentina, and compared some of its features with dengue presentation in the same region. METHODS: A retrospective study was conducted at San Vicente de Paul Hospital during 2016-2018. Clinical and demographic characteristics, pre-existing immunity to DENV, viral load and type I interferon (IFN) responses were studied in 63 patients with ZIKV infection. RESULTS: Clinical manifestations of ZIKV infection were generally mild compared with dengue, although rash (p<0.001) and itching (p<0.001) were significantly more prevalent in ZIKV patients. ZIKV patients aged <15 y manifested relatively mild disease compared with older ZIKV patients, showing a decreased prevalence of headache (p=0.008), retro-orbital pain (p=0.001) and arthralgia (p=0.001). Increased Zika incidence was observed in female patients (60.3%). Serum viral load was low to undetectable in ZIKV patients and was not associated with serum anti-DENV IgG titers. Interferon-α and IFN-ß serum levels did not correlate with serum viral load in ZIKV patients. CONCLUSIONS: Clinical presentation of ZIKV and DENV infections is largely overlapping, presenting a challenge for diagnosis and risk assessment for uniquely at-risk populations.

Murine models of dengue virus infection for novel drug discovery.

INTRODUCTION: Dengue virus (DENV) is the causative agent of the most prevalent human disease transmitted by mosquitoes in tropical and subtropical regions worldwide. At present, no antiviral drug is available and the difficulties to develop highly protective vaccines against the four DENV serotypes maintain the requirement of effective options for dengue chemotherapy. AREAS COVERED: The availability of animal models that reproduce human disease is a very valuable tool for the preclinical evaluation of potential antivirals. Here, the main murine models of dengue infection are described, including immunocompetent wild-type mice, immunocompromised mice deficient in diverse components of the interferon (IFN) pathway and humanized mice. The main findings in antiviral testing of DENV inhibitory compounds in murine models are also presented. EXPERT OPINION: At present, there is no murine model that fully recapitulates human disease. However, immunocompromised mice deficient in IFN-α/ß and -γ receptors, with their limitations, have shown to be the most suitable system for antiviral preclinical testing. In fact, the AG129 mouse model allowed the identification of celgosivir, an inhibitor of cellular glucosidases, as a promising option for DENV therapy. However, clinical trials still were not successful, emphasizing the difficulties in the transition from preclinical testing to human treatment.

A murine model of dengue virus infection in suckling C57BL/6 and BALB/c mice.

Dengue is a significant public health concern across tropical and subtropical regions worldwide, principally causing disease in children. Very young children are at increased risk of severe manifestations of dengue infection. The mechanism of dengue disease in this population is not fully understood. In this study, we present a murine model of dengue virus primary infection in suckling C57BL/6 and BALB/c mice in order to investigate disease pathogenesis. Three-day-old C57BL/6 mice intraperitoneally infected with DENV-2 NGC were more susceptible to infection than BALB/c mice, showing increased liver enzymes, extended viremia, dissemination to organs and histological alterations in liver and small intestine. Furthermore, the immune response in DENV-infected C57BL/6 mice exhibited a marked Th1 bias compared to BALB/c mice. These findings highlight the possibility of establishing an immunocompetent mouse model of DENV-2 infection in suckling mice that reproduces certain signs of disease observed in humans and that could be used to further study age-related mechanisms of dengue pathogenesis.

Role of the complement system in antibody-dependent enhancement of flavivirus infections.

Flavivirus infections have increased dramatically in the last decades in tropical and subtropical regions of the world. Antibody-dependent enhancement of dengue virus infections has been one of the main hypotheses to explain severity of disease and one of the major challenges to safe and effective vaccine development. In the presence of cross-reactive sub-neutralizing concentrations of anti-dengue antibodies, immune complexes can amplify viral infection in mononuclear phagocytic cells, triggering a cytokine cascade and activating the complement system that leads to severe disease. The complement system comprises a family of plasma and cellular surface proteins that recognize pathogen associated molecular patterns, modified ligands and immune complexes, interacting in a regulated manner and forming an enzymatic cascade. Pathogenic as well as protective effects of complement have been reported in flavivirus infections. This review provides updated knowledge on complement activation during flavivirus infection, including antiviral effects of complement and its regulation, as well as mechanisms of complement evasion and dysregulation of complement activity during viral infection leading to pathogenesis. Particularly, insights into classical pathway activation and its protective role on antibody-dependent enhancement of flavivirus infections are highlighted.

Human Type I Interferon Antiviral Effects in Respiratory and Reemerging Viral Infections.

Type I interferons (IFN-I) are a group of related proteins that help regulate the activity of the immune system and play a key role in host defense against viral infections. Upon infection, the IFN-I are rapidly secreted and induce a wide range of effects that not only act upon innate immune cells but also modulate the adaptive immune system. While IFN-I and many IFN stimulated genes are well-known for their protective antiviral role, recent studies have associated them with potential pathogenic functions. In this review, we summarize the current knowledge regarding the complex effects of human IFN-I responses in respiratory as well as reemerging flavivirus infections of public health significance and the molecular mechanisms by which viral proteins antagonize the establishment of an antiviral host defense. Antiviral effects and immune modulation of IFN-stimulated genes is discussed in resisting and controlling pathogens. Understanding the mechanisms of these processes will be crucial in determining how viral replication can be effectively controlled and in developing safe and effective vaccines and novel therapeutic strategies.

Interleukin-13 associates with life-threatening rhinovirus infections in infants and young children.

OBJECTIVE: Delineate risk factors associated with severe hypoxemia (O2 sat ≤87%) in infants and children younger than 2 years hospitalized with single pathogen HRV infection. STUDY DESIGN: Prospective study in a yearly catchment population of 56 560 children <2 years old between 2011 and 2013 in Argentina. All children with respiratory signs and O2 sat <93% on admission were included. HRV infections were identified by reverse transcriptase-polymerase chain reaction. Epidemiologic, clinical, viral, and immunological risk factors were assessed. RESULTS: Among 5012 hospitalized patients, HRV was detected as a single pathogen in 347 (6.92%) subjects. Thirty-two (9.2%) had life-threatening disease. Traditional risk factors for severe bronchiolitis did not affect severity of illness. HRV viral load, HRV groups, and type II and III interferons did not associate with severe hypoxemia. Interleukin-13 Levels in respiratory secretions at the time of admission (OR = 7.43 (3-18.4); P < 0.001 for IL-13 >10 pg/mL) predisposed to life-threatening disease. CONCLUSIONS: Targeted interventions against IL-13 should be evaluated to decrease severity of HRV illness in infancy and early childhood.

Age-associated differences in clinical manifestations and laboratory parameters during a dengue virus type 4 outbreak in Argentina.

Infection by any of the four dengue virus (DENV) serotypes produces a wide spectrum of clinical illness in humans. Differences in clinical manifestation and severity have been associated with secondary heterologous infection, patient age, and virus serotype. In this context, this retrospective study sought to analyze the presentation of dengue in patients during the 2014 DENV-4 outbreak affecting the City of Orán, Salta Province, Argentina. Demographic data, clinical manifestations, and laboratory abnormalities of laboratory-confirmed dengue patients were compared between age groups and between patients with and without warning signs. Of 301 patients with laboratory-confirmed dengue, 37.9% presented dengue with warning signs. Although nearly half of all patients had secondary DENV infections, no severe dengue cases, or deaths were reported. Furthermore, no association was found between incidence of warning signs and pre-existing immunity to DENV. Pediatric patients were least likely to present warning signs and showed significantly decreased risk of fever, retro-orbital pain, arthalgia, diarrhea and thrombocytopenia, and higher risk of rash compared to older patients. Female patients of all ages were also at higher risk of developing several symptoms. The characterization of DENV-4 infection in humans, a DENV serotype recently reported in Argentina, revealed differences in clinical manifestations, laboratory parameters and the presence/absence of warning signs based on age group. Further investigation of these age-related differences should contribute to better assessment of dengue disease in at risk populations.

Characterization of type I interferon responses in dengue and severe dengue in children in Paraguay.

BACKGROUND: Infection with dengue virus (DENV) produces a wide spectrum of clinical illness ranging from asymptomatic infection to mild febrile illness, and to severe forms of the disease. Type I interferons (IFNs) represent an initial and essential host defense response against viruses. DENV has been reported to trigger a robust type I IFN response; however, IFN-α/ß profile in the progression of disease is not well characterized. OBJECTIVES AND STUDY DESIGN: In this context, we conducted a retrospective study assessing the circulating serum levels of type I IFNs and related cytokines at different phases of illness in children during the 2011 outbreak of DENV in Paraguay. Demographic, clinical, laboratory and virological data were analyzed. RESULTS: During defervescence, significantly higher levels of IFN-ß, IL-6 and MIP-1ß, were detected in severe vs. non-severe dengue patients. Additionally, a significant positive correlation between INF-α and viremia was detected in children with severe dengue. A significant positive correlation was also observed between IFN-ß serum levels and hematocrit during the febrile phase, whereas IFN-α levels negatively correlated with white blood cells during defervescence in severe dengue patients. Furthermore, previous serologic status of patients to DENV did not influence type I IFN production. CONCLUSIONS: The distinct type I IFN profile in children with dengue and severe dengue, as well as its association with viral load, cytokine production and laboratory manifestations indicate differences in innate and adaptive immune responses that should be investigated further in order to unveil the association of immunological and physiological pathways that underlie in DENV infection.

The Role of Heterotypic DENV-specific CD8+T Lymphocytes in an Immunocompetent Mouse Model of Secondary Dengue Virus Infection.

Dengue is the most prevalent arthropod-borne viral disease worldwide and is caused by the four dengue virus serotypes (DENV-1-4). Sequential heterologous DENV infections can be associated with severe disease manifestations. Here, we present an immunocompetent mouse model of secondary DENV infection using non mouse-adapted DENV strains to investigate the pathogenesis of severe dengue disease. C57BL/6 mice infected sequentially with DENV-1 (strain Puerto Rico/94) and DENV-2 (strain Tonga/74) developed low platelet counts, internal hemorrhages, and increase of liver enzymes. Cross-reactive CD8+ T lymphocytes were found to be necessary and sufficient for signs of severe disease by adoptively transferring of DENV-1-immune CD8+T lymphocytes before DENV-2 challenge. Disease signs were associated with production of tumor necrosis factor (TNF)-α and elevated cytotoxicity displayed by heterotypic anti-DENV-1 CD8+ T lymphocytes. These findings highlight the critical role of heterotypic anti-DENV CD8+ T lymphocytes in manifestations of severe dengue disease.

Mortality due to Respiratory Syncytial Virus. Burden and Risk Factors.

RATIONALE: Respiratory syncytial virus (RSV) is the most frequent cause of hospitalization and an important cause of death in infants in the developing world. The relative contribution of social, biologic, and clinical risk factors to RSV mortality in low-income regions is unclear. OBJECTIVES: To determine the burden and risk factors for mortality due to RSV in a low-income population of 84,840 infants. METHODS: This was a prospective, population-based, cross-sectional, multicenter study conducted between 2011 and 2013. Hospitalizations and deaths due to severe lower respiratory tract illness (LRTI) were recorded during the RSV season. All-cause hospital deaths and community deaths were monitored. Risk factors for respiratory failure (RF) and mortality due to RSV were assessed using a hierarchical, logistic regression model. MEASUREMENTS AND MAIN RESULTS: A total of 2,588 (65.5%) infants with severe LRTI were infected with RSV. A total of 157 infants (148 postneonatal) experienced RF or died with RSV. RSV LRTI accounted for 57% fatal LRTI tested for the virus. A diagnosis of sepsis (odds ratio [OR], 17.03; 95% confidence interval [CI], 13.14-21.16 for RF) (OR, 119.39; 95% CI, 50.98-273.34 for death) and pneumothorax (OR, 17.15; 95% CI, 13.07-21.01 for RF) (OR, 65.49; 95% CI, 28.90-139.17 for death) were the main determinants of poor outcomes. CONCLUSIONS: RSV was the most frequent cause of mortality in low-income postneonatal infants. RF and death due to RSV LRTI, almost exclusively associated with prematurity and cardiopulmonary diseases in industrialized countries, primarily affect term infants in a developing world environment. Poor outcomes at hospitals are frequent and associated with the cooccurrence of bacterial sepsis and clinically significant pneumothoraxes.

Influenza and dengue virus co-infection impairs monocyte recruitment to the lung, increases dengue virus titers, and exacerbates pneumonia.

Co-infections of influenza virus and bacteria are known to cause severe disease, but little information exists on co-infections with other acute viruses. Seasonal influenza and dengue viruses (DENV) regularly co-circulate in tropical regions. The pandemic spread of influenza virus H1N1 (hereafter H1N1) in 2009 led to additional severe disease cases that were co-infected with DENV. Here, we investigated the impact of co-infection on immune responses and pathogenesis in a new mouse model. Co-infection of otherwise sublethal doses of a Nicaraguan clinical H1N1 isolate and two days later with a virulent DENV2 strain increased systemic DENV titers and caused 90% lethality. Lungs of co-infected mice carried both viruses, developed severe pneumonia, and expressed a unique pattern of host mRNAs, resembling only partial responses against infection with either virus alone. A large number of monocytes were recruited to DENV-infected but not to co-infected lungs, and depletion and adoptive transfer experiments revealed a beneficial role of monocytes. Our study shows that co-infection with influenza and DENV impairs host responses, which fail to control DENV titers and instead, induce severe lung damage. Further, our findings identify key inflammatory pathways and monocyte function as targets for future therapies that may limit immunopathology in co-infected patients.

Characterization of in vitro dengue virus resistance to carrageenan.

The λ-carrageenan (λ-car) is a potent and selective inhibitor of dengue virus (DENV) infection targeted to virus adsorption and internalization, due to the structural similarities with the mammalian cell receptor heparan sulfate. To further characterize the antiviral activity of λ-car, the selection and the phenotypic and genomic features of λ-car resistant DENV-2 variants are studied here in comparison to control virus. Resistant variants were rapidly selected in Vero cells after three passages in presence of the drug. No difference was detected in the growth profiles in Vero and C6/36 cells between resistant and control viruses. By contrast, the kinetics of adsorption and internalization of resistant variants in Vero cells was significantly diminished whereas entry to C6/36 cells was unaffected. By plaque purification and sequence analysis of the population, two types of resistant clones were found: some clones presented two mutations in E protein, K126E, and F422L; but other equally λ-car resistant clones had no mutations in E. Furthermore, no mutations were found in other viral proteins like prM, C, or NS1. The genomic disparity in E protein was also associated to differences in phenotype stability. The stable genomic resistance here described provides information about determinants in E protein involved in receptor binding and membrane fusion for uncoating.

Antiviral activity of an N-allyl acridone against dengue virus.

BACKGROUND: Dengue virus (DENV), a member of the family Flaviviridae, is at present the most widespread causative agent of a human viral disease transmitted by mosquitoes. Despite the increasing incidence of this pathogen, there are no antiviral drugs or vaccines currently available for treatment or prevention. In a previous screening assay, we identified a group of N-allyl acridones as effective virus inhibitors. Here, the antiviral activity and mode of action targeted to viral RNA replication of one of the most active DENV-2 inhibitors was further characterized. RESULTS: The compound 10-allyl-7-chloro-9(10H)-acridone, designated 3b, was active to inhibit the in vitro infection of Vero cells with the four DENV serotypes, with effective concentration 50% (EC50) values in the range 12.5-27.1 µM, as determined by virus yield inhibition assays. The compound was also effective in human HeLa cells. No cytotoxicity was detected at 3b concentrations up to 1000 µM. Mechanistic studies demonstrated that virus entry into the host cell was not affected, whereas viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR. The addition of exogenous guanosine together with 3b rescued only partially the infectivity of DENV-2. CONCLUSIONS: The acridone derivative 3b selectively inhibits the infection of Vero cells with the four DENV serotypes without a direct interaction with the host cell or the virion but interfering specifically with the intracellular virus multiplication. The mode of antiviral action for this acridone apparently involves the cellular enzyme inosine-monophospahe dehydrogenase together with another still unidentified target related to DENV RNA synthesis.

Changes in antiviral susceptibility to entry inhibitors and endocytic uptake of dengue-2 virus serially passaged in Vero or C6/36 cells.

The aim of the present study was to analyze the influence of virus origin, mammalian or mosquito cell-derived, on antiviral susceptibility of DENV-2 to entry inhibitors and the association of this effect with any alteration in the mode of entry into the cell. To this end, ten serial passages of DENV-2 were performed in mosquito C6/36 cells or monkey Vero cells and the antiviral susceptibility of each virus passage to sulfated polysaccharides (SPs), like heparin and carrageenans, was evaluated by a virus plaque reduction assay. After serial passaging in Vero cells, DENV-2 became increasingly resistant to SP inhibition whereas the antiviral susceptibility was not altered in virus propagated in C6/36 cells. The change in antiviral susceptibility was associated to a differential mode of entry into the host cell. The route of endocytic entry for productive Vero cell infection was altered from a non-classical clathrin independent pathway for C6/36-grown virus to a clathrin-mediated endocytosis when the virus was serially propagated in Vero cells. Our results show the impact of the cellular system used for successive propagation of DENV on the initial interaction between the host cell and the virion in the next round of infection and the relevant consequences it might have during the in vitro evaluation of entry inhibitors.

T helper type 2 bias and type 17 suppression in primary dengue virus infection in infants and young children.

BACKGROUND: The immune response to dengue virus (DENV) primary infection in infants and young children is not well characterized. In Northern Argentina, >90% of the population was DENV-naïve before the 2009 outbreak, allowing evaluation of age-dependent primary responses to infection. METHODS: We conducted a comparative study of the immune response to DENV in 27 infected infants, young children and their mothers. Lymphocyte T helper (Th) 1, Th2, Th17 and inflammatory responses were assayed in blood during the 2009 DENV-1 epidemic. RESULTS: The immune response to DENV-1 was significantly biased to Th2 in infected infants and young children, compared to infants with other febrile illnesses (for IL-4 p < 0.001) and to their infected mothers (for IL-4 p < 0.01). In addition, IL-17 suppression was observed in the memory response to DENV-1 in infected infants (p < 0.01 vs placebo). CONCLUSION: Age-related differences in the primary response to DENV, characterized by an immature Th2 polarization and Th17 suppression in infants, should be studied further in order to expand our understanding of the mechanism of dengue pathogenesis.

Differential inhibition of dengue virus infection in mammalian and mosquito cells by iota-carrageenan.

The antiviral activity against dengue virus-2 (DENV-2) of carrageenans reported here has shown a differential susceptibility of C6/36 HT and Vero cells, taken as models of mosquito and mammalian cells, depending on the structural class of polysaccharides: all polysaccharides blocked DENV-2 infection in monkey Vero cells, but only iota-carrageenans were virus inhibitors in mosquito cells. However, iota-carrageenans were less effective in mosquito cells in comparison with mammalian cells with effective concentration 50 % (EC(50)) values in C6/36 HT cells 4.9-17.5-fold higher than in Vero cells, as determined by virus yield reduction assay. The mode of action of iota-carrageenan in both cell types was strikingly different: in Vero cells the inhibitory activity was exerted only at the initiation of the cycle, affecting virion binding, whereas in mosquito cells DENV-2 adsorption was not affected and comparable levels of inhibition were obtained if the compound was added to cells together with the virus, after 8 h of infection or by cell pre-treatment before infection. Furthermore, iota-carrageenans induced a subtle alteration in mosquito cells, detected by cell proliferation and protein synthesis analyses, suggesting that a probable cellular target may be responsible for the refractory state of mosquito cells to DENV-2 infection produced by this class of polysulfates. The failure of iota-carrageenan to block DENV-2 adsorption to mosquito cells appeared to be related to the low presence of adequate heparan sulfate (HS) in C6/36 HT cell surface and is indicative of a differential participation of HS residues for DENV-2 entry in both types of cells.

Synthesis and antiviral activity of azoles obtained from carbohydrates.

Herein we describe the synthesis of 1,2,4-triazolyl-3-thione;1,3,4-oxadiazole, and imidazo[2,1-b]thiazole derivatives from carbohydrates. The antiviral activity of these compounds was tested against Dengue and Junin virus (the etiological agent of Argentine hemorrhagic fever). The 3-(p-bromobenzoyl)-5-(1,2-O-isopropylidene-3-O-methyl-alpha-d-xylofuranos-5-ulos-5-yl)imidazo[2,1-b]thiazole was able to inhibit the replication of both viruses in Vero cells at concentration significantly lower than the CC(50).

An algal-derived DL-galactan hybrid is an efficient preventing agent for in vitro dengue virus infection.

The DL-galactan hybrid C2S-3, isolated from the red seaweed Cryptonemia crenulata (Halymeniaceae, Halymeniales), is a potent and selective inhibitor of the multiplication of diverse strains of DENV-2 in Vero cells with higher effectiveness than the reference polysaccharide heparin. The presence of the compound either only at virus adsorption or at virus internalization exerted a significant and dose-dependent inhibition in DENV-2 plaque number. The compound failed to inactivate DENV-2 directly by incubation of virus before cell infection as well as to induce a refractory state by cell pretreatment. Thus, the inhibitory effect was exclusively exerted through a blockade in virus multiplication during the infectious process. When the entry of DENV-2 particles into the cell is bypassed, as occurs in virus RNA transfection, the polysaccharide C2S-3 failed to block the completion of the multiplication cycle. Furthermore, the antiviral properties of C2S-3 are not correlated with anticoagulant activity.

Interference in dengue virus adsorption and uncoating by carrageenans.

This study demonstrated that the lambda- and iota-carrageenans, sulfated polysaccharides containing linear chains of galactopyranosyl residues, are potent inhibitors of dengue virus type 2 (DENV-2) and 3 (DENV-3) multiplication in Vero and HepG2 cells, with values of effective concentration 50% from 0.14 to 4.1 microg/ml. This activity was assayed by plaque reduction, virus yield inhibition and antigen expression tests, and was independent of the input multiplicity of infection in the range 0.001-1. The inhibitory action of the lambda-carrageenan, an heparan sulfate (HS)-imitative compound, was exerted by a dual interference with virus adsorption and internalization of nucleocapsid into the cytoplasm. Although virus particles may enter the cell when compound was added after DENV-2 adsorption, as shown by intracellular uptake of radiolabeled DENV-2 particles and quantitative RT-PCR, infectious center and virion uncoating assays have shown that carrageenan-treated virions cannot be released from the endosomes. Viral protein synthesis, the first step of macromolecular synthesis after DENV entry to the host cell, was not affected by the carrageenan. Furthermore, no inhibition of virus multiplication was detected when the entry process was bypassed through DENV-2 RNA transfection into the cell. The dual sites of action of an HS-like molecule suggest that, at least in monkey kidney and human hepatic cells, the HS residues in the cell membrane appear to act as mediators for DENV-2 entry, an interesting alternative target for flavivirus therapy.