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Severe coronavirus disease 2019 (COVID-19) is characterized by lung injury, cytokine storm, and increased neutrophil-to-lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe COVID-19 in infected individuals. Angiotensin-converting enzyme-2 (ACE2) is the receptor for SARS-CoV-2, the virus causing COVID-19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates COVID-19-related lung inflammation. We employed a preclinical mouse model using intratracheal instillation of a combination of polyinosinic:polycytidylic acid (poly(I:C)) and the receptor-binding domain of the SARS-CoV-2 spike protein (RBD-S) to mimic lung damage associated with COVID-19. Histologic analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion, and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill COVID-19 patients. Administration of the ADAM17/MMP inhibitors apratastat and TMI-1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of proinflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM-1 and VCAM-1. Additionally, the NLR was significantly reduced by ADAM17/MMP inhibition. Thus, we propose inhibition of ADAM17/MMP as a novel promising treatment strategy in SARS-CoV-2-infected individuals to prevent the progression toward severe COVID-19.

International audience; The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.

Severe coronavirus disease 2019 (Covid-19) is characterized by lung injury, cytokine storm and increased neutrophil-to-lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe Covid-19 in infected individuals. Angiotensin-converting enzyme-2 ACE-2) is the receptor for SARS-CoV-2, the virus causing Covid-19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE-2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates Covid-19-related lung inflammation. We employed a pre-clinical mouse model using intra-tracheal instillation of a combination of polyinosinic:polycytidylic acid (poly-I:C) and the receptor-binding domain of the SARS-CoV-2 spike protein (RBD-S) to mimic lung damage associated with Covid-19. Histological analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill Covid-19 patients. Administration of the ADAM17 inhibitors apratastat and TMI-1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of pro-inflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM-1 and VCAM-1. Additionally, the NLR was significantly reduced by ADAM17 inhibition. Thus, we propose inhibition of ADAM17 as a novel promising treatment strategy in SARS-CoV-2-infected individuals to prevent the progression towards severe Covid-19.

Dengue is a worldwide expanding threat caused by dengue virus (DENV) infection. To date, no specific treatment or effective vaccine is available. Antibodies produced by plasma cells (PCs) might be involved concomitantly in protection and severe dengue immunopathology. Although a massive appearance of PCs has been reported during acute DENV infection in humans, this response has been poorly characterized. Here, we show the dynamic of PC generation in immune-competent mice cutaneously inoculated with DENV compared with two control experimental groups: mice inoculated with inactivated DENV or with PBS. We found that PC numbers increased significantly in the skin-draining lymph node (DLN), peaking at day 10 and abruptly decreasing by day 14 after DENV inoculation. Class-switched IgG+ PCs appeared from day 7 and dominated the response, while in contrast, the frequency of IgM+ PCs decreased from day 7 onwards. Even though the kinetic of the response was similar between DENV- and iDENV-inoculated mice, the intensity of the response was significantly different. Interestingly, we demonstrated a similar PC response to virus antigens (E and prM) by ELISPOT. In situ characterization showed that PCs were distributed in the medullary cords and in close proximity to germinal centers (GCs), suggesting both an extrafollicular and a GC origin. Proliferating PCs (Ki-67+) were found as early as 3-day postinoculation, and in-depth analysis showed that these PCs were in active phases of cell cycle during the kinetic. Finally, we found a progressive appearance of high-affinity neutralizing DENV-specific IgG further supporting GC involvement. Of note, these antibodies seem to be highly cross-reactive, as a large proportion recognizes Zika virus (ZIKV). The strong PC response to skin-inoculated DENV in this work resembles the findings already described in humans. We consider that this study contributes to the understanding of the in vivo biology of the humoral immune response to DENV in an immunocompetent murine model.

Dengue virus infection (DENV-2) is transmitted by infected mosquitoes via the skin, where many dermal and epidermal cells are potentially susceptible to infection. Most of the cells in an area of infection will establish an antiviral microenvironment to control viral replication. Although cumulative studies report permissive DENV-2 infection in dendritic cells, keratinocytes, and fibroblasts, among other cells also infected, little information is available regarding cell-to-cell crosstalk and the effect of this on the outcome of the infection. Therefore, our study focused on understanding the contribution of fibroblast and dendritic cell crosstalk to the control or promotion of dengue. Our results suggest that dendritic cells promote an antiviral state over fibroblasts by enhancing the production of type I interferon, but not proinflammatory cytokines. Infected and non-infected fibroblasts promoted partial dendritic cell maturation, and the fibroblast-matured cells were less permissive to infection and showed enhanced type I interferon production. We also observed that the soluble mediators produced by non-infected or Poly (I:C) transfected fibroblasts induced allogenic T cell proliferation, but mediators produced by DENV-2 infected fibroblasts inhibited this phenomenon. Additionally, the effects of fibroblast soluble mediators on CD14+ monocytes were analyzed to assess whether they affected the differentiation of monocyte derived dendritic cells (moDC). Our data showed that mediators produced by infected fibroblasts induced variable levels of monocyte differentiation into dendritic cells, even in the presence of recombinant GM-CSF and IL-4. Cells with dendritic cell-like morphology appeared in the culture; however, flow cytometry analysis showed that the mediators did not fully downregulate CD14 nor did they upregulate CD1a. Our data revealed that fibroblast-dendritic cell crosstalk promoted an antiviral response mediated manly by type I interferons over fibroblasts. Furthermore, the maturation of dendritic cells and T cell proliferation were promoted, which was inhibited by DENV-2-induced mediators. Together, our results suggest that activation of the adaptive immune response is influenced by the crosstalk of skin resident cells and the intensity of innate immune responses established in the microenvironment of the infected skin.

The skin is the first anatomical region that dengue virus (DENV) encounters during the natural infection. Although the role of some skin resident cells like dendritic cells and fibroblasts has been demonstrated to be crucial to elucidate the role of resident cells and molecules participating during the early events of the innate immune response, the participation of keratinocytes during DENV infection has not been fully elucidated. In this paper we aimed to evaluate the use of the HaCaT cell line as a model to study the immune responses of skin keratinocytes to DENV infection. We demonstrated productive DENV-2 infection of HaCaT cells and their capability to establish an antiviral response through production of type I and type III interferons (IFN-β and IFN-λ). The production of these cytokines by HaCaT cells correlated with upregulation of IFN-inducible transmembrane protein-3 (IFITM3) and viperin in bystander, uninfected cells. We also observed an increase in secretion of IL-6 and IL-8. Skin keratinocytes are known to secrete antimicrobial peptides (AMPs) during viral infections. In our model, DENV-2 infected HaCaT cells upregulate the production of cytoplasmic LL-37. We evaluated the dual role of LL-37, HBD2, and HBD3 antiviral activity and immunoregulation during DENV-2 infection of HaCaT cells and found that LL-37 significantly reduced DENV-2 replication. This indicates that the HaCaT cell line can be used as a model for studying the innate response of keratinocytes to DENV infection. Our results also suggest that skin keratinocytes play an important role in the skin microenvironment after DENV infection by secreting molecules like type I and type III IFNs, pro-inflammatory molecules, and LL-37, which may contribute to the protection against arboviral infections.

Background Co-circulation of dengue virus (DENV) and chikungunya virus (CHIKV) is increasing worldwide but information on the viral dynamics and immune response to DENV-CHIKV co-infection, particularly in young infants, is scant. Methods Blood samples were collected from 24 patients, aged 2 months to 82 years, during a CHIKV outbreak in Mexico. DENV and CHIKV were identified by RT-PCR; ELISA was used to detect IgM and IgG antibodies. CHIKV PCR products were cloned, sequenced and subjected to BLAST analysis. To address serological findings, HMEC-1 and Vero cells were inoculated with DENV-1, DENV-2 and CHIKV alone and in combination (DENV-2-CHIKV and DENV-1-CHIKV); viral titers were measured at 24, 48 and 72 h. Results Nine patients (38%) presented co-infection, of who eight were children. None of the patients presented severe illness. Sequence analysis showed that the circulating CHIKV virus belonged to the Asian lineage. Seroconversion to both viruses was only observed in the four patients five years or older, while the five infants under two years of age only seroconverted to CHIKV. Viral titers in the CHIKV mono-infected cells were greater than in the DENV-1 and DENV-2 mono-infected cells. Furthermore, we observed significantly increased CHIKV progeny and reduction of DENV progeny in the co-infected cells. Conclusions In our population, DENV-CHIKV co-infection was not associated with increased clinical severity. Our in vitro assay findings strongly suggest that the lack of DENV IgG conversion in the co-infected infants is due to suppression of DENV replication by the Asian lineage CHIKV. The presence of maternal antibody and immature immune responses in the young infants may also play a role. Electronic supplementary material The online version of this article (10.1186/s13071-018-2942-1) contains supplementary material, which is available to authorized users.

During infection with Dengue virus (DENV), host cells undergo a series of detrimental functional changes, including profound remodelling and redistribution of cell membrane structures,1,4 which giv...

Dengue is a global health problem without current specific treatment nor safe vaccines available. While severe dengue is related to pre-existing non-neutralizing dengue virus (DENV) antibodies, the role of T cells in protection or pathology is unclear. Using cutaneous DENV infection in immunocompetent mice we previously showed the generation of PNA+ germinal centers (GCs), now we assessed the activation and proliferation of B and T cells in draining lymph nodes (DLNs). We found a drastic remodelling of DLN compartments from 7 to 14 days post-infection (dpi) with greatly enlarged B cell follicles, occupying almost half of the DLN area compared to ~24% in naive conditions. Enormous clusters of proliferating (Ki-67+) cells inside B follicles were found 14 dpi, representing ~33% of B cells in DLNs but only ~2% in non-infected mice. Inside GCs, we noticed an important recruitment of tingle body macrophages removing apoptotic cells. In contrast, the percentage of paracortex area and total T cells decreased by 14–16 dpi, compared to controls. Scattered randomly distributed Ki-67+ T cells were found, similar to non-infected mice. CD69 expression by CD4+ and CD8+ T cells was minor, while it was remarkable in B cells, representing 1764.7% of change from basal levels 3 dpi. The apparent lack of T cell responses cannot be attributed to apoptosis since no significant differences were observed compared to non-infected mice. This study shows massive B cell activation and proliferation in DLNs upon DENV infection. In contrast, we found very poor, almost absent CD4+ and CD8+ T cell responses.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has caused recent large outbreaks in the Americas. Given its association with severe congenital defects including microcephaly, distinguishing infections caused by ZIKV from those caused by dengue virus (DENV) is of primordial importance. The objectives of this study were to evaluate the recombinant proteins rEIII-ZIKV (Envelope protein domain III) and rNS1s-leader-ZIKV (non-structural protein 1) for the serological diagnosis of ZIKV in the Mexican population. We also evaluated potential cross-reactivity in commercial enzyme-linked immunosorbent assays (ELISA) based on the ZIKV NS1 and DENV NS1 proteins. rEIII-ZIKV and rNS1s-leader-ZIKV proteins were tested with sera from 30 PCR-confirmed ZIKV cases, 50 ZIKV-naive, DENV-exposed subjects with no acute febrile disease, (asymptomatic subjects, AS), and 50 ZIKV-naive and DENV naive AS. Commercial ELISA tests were evaluated with sera from 57 ZIKV and 20 DENV PCR-confirmed cases, and 50 ZIKV-naive, DENV-exposed AS. In-house ELISA assays showed that IgM antibody levels against rEIII-ZIKV and rNS1s-ZIKV were higher in ZIKV naive, DENV-exposed AS than in acutely infected ZIKV individuals. IgG reactivity was highest for rEIII-ZIKV, and indistinguishable between acutely infected ZIKV cases and DENV exposed AS. Positivity for the Euroimmun Zika IgM assay at 7-10 days was considerably higher in DENV-naive ZIKV patients (86%) than in DENV-exposed ZIKV patients (33%), while 39% of the latter had false-negative anti-ZIKV IgG before 7 days of onset. DENV-exposed ZIKV patients presented lower anti-ZIKV IgM and higher IgG responses similar to a secondary dengue response. Forty-four percent of DENV- exposed acute ZIKV patients were DENV IgM positive with the Panbio Dengue assay, and two (15%) of the DENV-naive ZIKV patients presented false DENV IgG conversion. Given the extensive cross-reactivity to both the NS1 and EDIII proteins in current serological methods, the development of sensitive and specific serological tests to distinguish ZIKV from DENV infections is an urgent priority.

The envelope (E) protein from Dengue and Zika viruses comprises three functional and structural domains (DI, DII, and DIII). Domain III induces most of the neutralizing antibodies and, as such, is considered as having the highest antigenic potential for the evaluation of population-level surveillance and for detecting past infections in both Dengue and Zika patients. The present study aimed to clone and express recombinant proteins of domain III from Dengue virus serotype 2 and from Zika virus in a prokaryotic system, as well as evaluate their immunogenicity and cross-reactivity. Both antigens were successfully purified and their antigenicity was assessed in mice. The antibodies elicited by domain III of Zika and Dengue virus antigens recognized specifically the native proteins in infected cells. Furthermore, the antigens showed a more specific immunogenic response than that of domain III proteins from Dengue virus. The generated recombinant proteins can be potentially used in subunit vaccines or for surveillance studies.

Zika virus (ZIKV), an emerging mosquito-borne flavivirus, has quickly spread in many regions around the world where dengue virus (DENV) is endemic. This represents a major health concern, given the high homology between these two viruses, which can result in cross-reactivity. The aim of this study was to determine the cross-reacting antibody response of the IgM and IgG classes against the recombinant envelope protein of ZIKV (rE-ZIKV) in sera from patients with acute-phase infection of different clinical forms of dengue, i.e., dengue fever (DF) and dengue hemorrhagic fever (DHF) (before the arrival of ZIKV in Mexico 2010), as well as acute-phase sera of ZIKV patients, together with the implications in neutralization and antibody-dependent enhancement. Differences in IgM responses were observed in a number of DF and DHF patients whose sera cross-reacted with the rE-ZIK antigen, with 42% recognition between acute-phase DHF and ZIKV but 27% recognition between DF and ZIKV. Regarding IgG antibodies, 71.5% from the DF group showed cross-reactivity to rE-ZIKV in contrast with 50% and only 25% of DHF and ZIKV serum samples, respectively, which specifically recognized the homologous antigen. The DHF group showed more enhancement of ZIKV infection of FCRγ-expressing cells compared to the DF group. Furthermore, the DHF group also showed a higher cross-neutralizing ability than that of DF. This is the first report where DF and DHF serum samples were evaluated for cross-reactivity against Zika protein and ZIKV. Furthermore, DENV serum samples cross-protect against ZIKV through neutralizing antibodies but at the same time mediate antibody-dependent enhancement in the sequential ZIKV infection.

The respiratory syncytial virus (RSV) is the main pathogen associated with upper respiratory tract infections during early childhood. Vertical transmission of this virus has been suggested in humans, based on observations recorded during animal studies that revealed an association of RSV with persistent structural and functional changes in the developing lungs of the offspring. However, human placentas have not yet been evaluated for susceptibility to RSV infection. In this study, we examined the capacity of RSV to infect a human trophoblast model, the BeWo cell line. Our results suggest that BeWo cells are susceptible to RSV infection since they allow RNA viral replication, viral protein translation, leading to the production of infectious RSV particles. In this report, we demonstrate that a human placenta model system, consisting of BeWo cells, is permissive to RSV infection. Thus, the BeWo cell line may represent a useful model for studies that aim to characterize the events of a possible RSV infection at the human maternal–fetal interface.

Flaviviruses are positive, single-stranded, enveloped cytoplasmic sense RNA viruses that cause a variety of important diseases worldwide. Among them, Zika virus, West Nile virus, Japanese encephalitis virus, and Dengue virus have the potential to cause severe disease. Extensive studies have been performed to elucidate the structure and replication strategies of flaviviruses, and current studies are aiming to unravel the complex molecular interactions between the virus and host during the very early stages of infection. The outcomes of viral infection and rapid establishment of the antiviral state, depends on viral detection by pathogen recognition receptors and rapid initiation of signalling cascades to induce an effective innate immune response. Extracellular and intracellular pathogen recognition receptors play a crucial role in detecting flavivirus infection and inducing a robust antiviral response. One of the main hallmarks of flaviviral nonstructural proteins is their multiple strategies to antagonise the interferon system. In this chapter, we summarize the molecular characteristics of flaviviral proteins and discuss how viral proteins target different components of the interferon signalling pathway by blocking phosphorylation, enhancing degradation, and downregulating the expression of major components of the Janus kinase/signal transducer and activator of transcription pathway. We also discuss how the interactions of viral proteins with host proteins facilitate viral pathogenesis. Due to the lack of antivirals or prophylactic treatments for many flaviviral infections, it is necessary to fully elucidate how these viruses disrupt cellular processes to influence pathogenesis and disease outcomes.

The placenta is a highly specialized organ that is formed during human gestation for conferring protection and generating an optimal microenvironment to maintain the equilibrium between immunological and biochemical factors for fetal development. Diverse pathogens, including viruses, can infect several cellular components of the placenta, such as trophoblasts, syncytiotrophoblasts and other hematopoietic cells. Viral infections during pregnancy have been associated with fetal malformation and pregnancy complications such as preterm labor. In this minireview, we describe the most recent findings regarding virus–host interactions at the placental interface and investigate the mechanisms through which viruses may access trophoblasts and the pathogenic processes involved in viral dissemination at the maternal–fetal interface., One current overview about viral entry and dissemination mechanism at the maternal–fetal interface.

Dengue virus (DENV) is the causal agent of severe disease and, in some cases, mortality in humans, but no licensed vaccines against dengue are available. An effective vaccine against dengue requires long-term humoral and cellular immune responses. Several viral proteins have been the subjects of intense research, especially the envelope (E) protein, aimed at developing a vaccine. Domain III of the envelope protein (EDIII) has been identified as a potential candidate because it is involved in binding to host cell receptors and contains epitopes that elicit virus neutralizing antibodies. However, this domain is not sufficiently antigenic when is expressed and administered as antigen to elicit a strong immune response. One alternative to enhance immunogenicity is to target the antigen to dendritic cells to induce T-cells for broad antibody responses. In this work, a single chain antibody fragment (scFv) raised against the DEC-205 receptor fused with the EDIII was successfully expressed in Nicotiana benthamiana. The recombinant protein was expressed and purified from the plant and evaluated in BALB/c mice to test its immunogenicity and ability to induce neutralizing antibodies against DENV. The mice immunized with the recombinant protein produced specific and strong humoral immune responses to DENV. Only two immunizations were required to generate a memory response to DENV without the presence of adjuvants. Also, recognition of the recombinant protein with sera from DENV-infected patients was observed. These findings suggest that this strategy has potential for development of an effective vaccine against DENV.

Dengue viruses (DENVs; serotypes 1-4) are members of the flavivirus family. The envelope protein (E) of DENV has been defined as the principal antigenic target in terms of protection and diagnosis. Antibodies that can reliably detect the E surface glycoprotein are necessary for describing and mapping new DENV epitopes as well as for developing more reliable and inexpensive diagnostic assays. In this study, we describe the production and characterization of a monoclonal antibody (mAb) against a recombinant DENV-2 E protein that recognizes a sequential antigen in both native and recombinant form located in domain II of the E protein of all four DENV serotypes. We confirmed that this mAb, C21, recognizes a sequence located in the fusion peptide. In addition, C21 does not have neutralizing activity against DENV-2 in an in vitro system. Furthermore, the C21 mAb is an ideal candidate for the development of research reagents for studying DENV biology because it cross-reacts with the four dengue serotypes.

A complex interplay between pathogen and host determines the immune response during viral infection. A set of cytosolic sensors are expressed by immune cells to detect viral infection. NOD-like receptors (NLRs) comprise a large family of intracellular pattern recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed inflammasomes, which induce downstream immune responses to specific pathogens, environmental stimuli, and host cell damage. Inflammasomes are composed of cytoplasmic sensor molecules such as NLRP3 or absent in melanoma 2 (AIM2), the adaptor protein ASC (apoptosis-associated speck-like protein containing caspase recruitment domain), and the effector protein procaspase-1. The inflammasome operates as a platform for caspase-1 activation, resulting in caspase-1-dependent proteolytic maturation and secretion of interleukin (IL)-1β and IL-18. This, in turn, activates the expression of other immune genes and facilitates lymphocyte recruitment to the site of primary infection, thereby controlling invading pathogens. Moreover, inflammasomes counter viral replication and remove infected immune cells through an inflammatory cell death, program termed as pyroptosis. As a countermeasure, viral pathogens have evolved virulence factors to antagonise inflammasome pathways. In this review, we discuss the role of inflammasomes in sensing viral infection as well as the evasion strategies that viruses have developed to evade inflammasome-dependent immune responses. This information summarises our understanding of host defence mechanisms against viruses and highlights research areas that can provide new approaches to interfere in the pathogenesis of viral diseases.

When dengue virus (DENV)-infected mosquitoes use their proboscis to probe into human skin during blood feeding, both saliva and virus are released. During this process, cells from the epidermis and dermis layers of the skin, along with small blood vessels, may get exposed to or infected with DENV. In these microenvironments of the skin, the presence of DENV initiates a complex interplay among the DENV-infected and non-infected neighboring cells at the initial bite site. Previous studies suggested that DENV-infected human dermal fibroblasts (HDFs) participate in the immune response against DENV by secreting soluble mediators of innate immunity. In the present study, we investigated whether DENV-infected HDFs activate human dermal microvascular endothelial cells (HDMECs) in co-cultures. Our results suggest that co-cultures of DENV-infected HDFs and HDMECs elicit soluble mediators that are sufficient to reduce viral replication, activate HDMECs, and induce leukocyte migration through HDMEC monolayers. These effects were partly dependent on HDF donor and DENV serotype, which may provide novel insights into the natural variation in host susceptibility to DENV disease.

Dengue virus (DENV) has four serotypes, which can cause from asymptomatic disease to severe dengue. Heterologous secondary infections have been associated to a greater risk of potentially fatal dengue due to non-neutralizing memory antibodies (Abs), which facilitate the infection, such as anti-precursor membrane (prM) Abs, among other mechanisms. Usually, class-switched memory Abs are generated mainly through germinal centers (GCs). However, the cellular events underlying these Ab responses to DENV, especially during repeated/secondary infections, have been poorly studied. We wanted to know whether there is involvement of GC reactions during cutaneous DENV infection and whether there is any sort of preferential Ab responses to defined viral proteins. Intradermal DENV inoculation at a relatively low dose efficiently infects immune-competent BALB/c mice, inducing higher quantities of DENV-specific GC B cells and larger GCs than the control conditions. Interestingly, GCs exhibited as much prM as envelope (E) and non-structural 3 viral proteins in situ. Intriguingly, despite the much larger abundance of E protein than of prM protein in the virions, infected animals showed similar amounts of circulating Abs and Ag-specific GC B cells both for prM and for E proteins, even significantly higher for prM. To the best of our knowledge, there are no reports of the GC responses during DENV infection. This relatively stronger anti-prM response could be triggered by DENV to preferentially promote Abs against certain viral proteins, which might favor infections by facilitating DENV invasion of host cells. It is thus conceivably that DENV might have evolved to induce this kind of Ab responses.

Recently, Man et al . [2016] published an elegant study in which they showed that immunity-related GTPase family member b10 (IRGB10; an interferon-inducible protein) localizes on the cell membrane of bacteria, damages the membrane, and then releases the hidden ligands from the phagosome to the host-cytoplasm to be sensed by both the absent in melanoma 2 (AIM2) and the noncanonical NLR family pyrin domain-containing (NLRP) inflammasomes (1).

In this review, we discuss the current knowledge of the role of the antibody response against dengue virus and highlight novel insights into targets recognized by the human antibody response. We also discuss how the balance of pathological and protective antibody responses in the host critically influences clinical aspects of the disease.

Dengue virus (DENV) RNA replication requires 2 viral proteins, non-structural protein 3 (NS3) and NS5. NS5 consists of 2 functional domains: a methyltransferase (MTase) domain involved in RNA cap formation and located in the amino terminal region and a RNA-dependent RNA polymerase domain essential for virus replication and located in the carboxyl terminal region. To gain additional insight into the structural interactions between viral proteins and cellular factors involved in DENV RNA replication, we generated a panel of rat monoclonal antibodies (mAbs) against the NS5 MTase domain. Six rat mAbs were selected from 41 clones, of which clone 13G7 was further characterized. The specificity of this antibody for NS5 was demonstrated by western blot of DENV-infected cells, which revealed that this antibody recognizes all 4 DENV serotypes. Furthermore, Western blotting analysis suggested that this antibody recognizes a sequential epitope of the NS5 protein. Positive and specific staining with 13G7 was detected predominantly in nuclei of DENV-infected cells, similarly a pattern was observed in both in human and monkey cells. Furthermore, the NS5 staining co-localized with a Lamin A protein (Pierson index: 0.7). In summary, this monoclonal antibody could be used to identify and evaluate different cellular factors that may interact with NS5 during DENV replication.

A DENV-2 plasmid named pEII*EIII/NS1*, containing sequences encoding portions of the envelope protein that are potentially involved in the induction of neutralizing antibodies and a portion of the NS1 sequence that is involved in protection, is reported in this work. The synthesized subunit protein was recognized by human sera from infected patients and had the predicted size. The immunogenicity of this construct was evaluated using a mouse model in a prime-boost vaccination approach. The priming was performed using the plasmid pEII*EIII/NS1*, followed by a boost with recombinant full-length GST–E and GST–NS1 fusion proteins. The mice showed specific antibody responses to the E and NS1 proteins, as detected by ELISA, compared to the response of animals vaccinated with the parental plasmid. Interestingly, some animals had neutralizing antibodies. These results show that EII*, EIII and NS1* sequences could be considered for the design of a recombinant subunit vaccine against dengue disease.

This study was undertaken to evaluate the feasibility of using recombinant dengue proteins to discriminate between acute dengue infections versus uninfected dengue samples. Dengue virus proteins E, NS1, NS3, and NS4B were cloned as fusion proteins and expressed in Escherichia coli. Recombinant products were tested in 100 serum samples obtained from acute dengue fever cases collected from 3 states of Mexico where dengue is endemic. Sera from 75 healthy individuals living in nonendemic areas for dengue were used as a control group. In sera from the dengue patients group, antibody responses to E protein were demonstrated in 91% of cases and NS1 protein was recognized to various extents (99%) within the first 7 days of infection. The antibody responses to NS3 and NS4B were frequently of low magnitude. Consistent negative antibody responses to all proteins were found in sera from the control group. These data suggest that the glutathione-S-transferase (GST)-dengue fusion proteins may be feasible antigens for a sensitive and specific serological assay.

DNA priming-protein boosting is a strategy used to establish strong immunity to a specific pathogen by the use of two different antigens through sequential delivery systems. In this work, two recombinant plasmids were used, one encoding for the dengue virus E protein, which is know to induce neutralizing antibodies (pcDNA 3.1/E), and the other encoding for the Dengue virus nonstructural protein 1 (pcDNA 3.1/NS1), as a source of B- and T-cell epitopes possibly involved in protective immunity. We showed that immunization of BALB/c mice with three priming doses of both plasmids pcDNA 3.1/E and/or pcDNA 3.1/NS1 were able to induce antibody responses to E protein with a single plasmid; in contrast to the antibody response to NS1 protein we observed an additive effect in terms of antibody response. Moreover, using a prime-boost protocol in which both plasmid constructs were co-administrated followed by a boost of homologous GST-E and GST-NS1 recombinant proteins, we observed an increased antibody response to NS1 and to E protein compared to animals vaccinated with the proteins or with dengue constructs alone. If neutralizing antibodies play an important role in dengue infection, antibodies generated with this regimen was also significantly better than the administration of the mix of proteins alone. These results suggest that NS1 and E proteins together could be considered in a design of subunit recombinant vaccines.

Dengue is considered a reemerging disease of worldwide distribution. The Dengue virus non-structural protein 3 (NS3) is known to possess ATPase, helicase, and protease activities that are a constitutive part of the replication complex of Dengue virus. In this report, we discuss the cloning, expressing, and purifying of the Dengue-2 NS3 protein, to immunize mice and then generate monoclonal antibodies (MAbs). Our results show the production of MAbs specific to NS3 protein of Dengue-2 virus, which by immunofluorescence recognize the native protein in experimentally infected endothelial cells (HMEC). Likewise, C6/36-infected lisates were used in Western blots, and observed the specific characteristic band that defines the NS3 protein. We conclude that these antibodies may be a useful tool, not only to study the replicative process of Dengue virus, but also to generate specific diagnostic tools for Dengue infection.

Lipid rafts are ordered microdomains within cellular membranes that are rich in cholesterol and sphingolipids. Caveolin (Cav-1) and flotillin (Flt-1) are markers of lipid rafts, which serve as an organizing center for biological phenomena and cellular signaling. Lipid rafts involvement in dengue virus (DENV) processing, replication, and assembly remains poorly characterized. Here, we investigated the role of lipid rafts after DENV endocytosis in human microvascular endothelial cells (HMEC-1). The non-structural viral proteins NS3 and NS2B, but not NS5, were associated with detergent-resistant membranes. In sucrose gradients, both NS3 and NS2B proteins appeared in Cav-1 and Flt-1 rich fractions. Additionally, double immunofluorescence staining of DENV-infected HMEC-1 cells showed that NS3 and NS2B, but not NS5, colocalized with Cav-1 and Flt-1. Furthermore, in HMEC-1cells transfected with NS3 protease, shown a strong overlap between NS3 and Cav-1, similar to that in DENV-infected cells. In contrast, double-stranded viral RNA (dsRNA) overlapped weakly with Cav-1 and Flt-1. Given these results, we investigated whether Cav-1 directly interacted with NS3. Cav-1 and NS3 co-immunoprecipitated, indicating that they resided within the same complex. Furthermore, when cellular cholesterol was depleted by methyl-beta cyclodextrin treatment after DENV entrance, lipid rafts were disrupted, NS3 protein level was reduced, besides Cav-1 and NS3 were displaced to fractions 9 and 10 in sucrose gradient analysis, and we observed a dramatically reduction of DENV particles release. These data demonstrate the essential role of caveolar cholesterol-rich lipid raft microdomains in DENV polyprotein processing and replication during the late stages of the DENV life cycle.

Background When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times (“probing”) before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells. Methodology/Principal Findings Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 (IRF7), when compared with mock-infected fibroblasts. Conclusions/Significance In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and provide viral particles that may contribute to subsequent viral dissemination., Author Summary In this work, we demonstrate that that both human whole skin and freshly isolated skin fibroblasts are productively infected with Dengue virus (DENV). In addition, primary skin fibroblast cultures were established and subsequently infected with DENV-2; we showed in these cells the presence of the viral antigen NS3, and we found productive viral infection by a conventional plaque assay. Of note, the infectivity rate was almost the same in all the primary cultures analyzed from different donors. The skin fibroblasts infected with DENV-2 underwent signaling through both TLR3 and RIG-1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 IRF7, when compared with mock-infected fibroblasts. Our data suggest that fibroblasts might even participate producing mediators involved in innate immunity that activate and contribute to the orchestration of the local innate responses. This work is the first evaluating primary skin fibroblast cultures obtained from different humans, assessing both their susceptibility to DENV infection as well as their ability to produce molecules crucial for innate immunity.

Background One of the main phenomena occurring in cellular membranes during virus infection is a change in membrane permeability. It has been observed that numerous viral proteins can oligomerize and form structures known as viroporins that alter the permeability of membranes. Previous findings have identified such proteins in cells infected with Japanese encephalitis virus (JEV), a member of the same family that Dengue virus (DENV) belongs to (Flaviviridae). In the present work, we investigated whether the small hydrophobic DENV protein NS2B serves a viroporin function. Methods We cloned the DENV NS2B sequence and expressed it in a bacterial expression system. Subsequently, we evaluated the effect of DENV NS2B on membranes when NS2B was overexpressed, measured bacterial growth restriction, and evaluated changes of permeability to hygromycin. The NS2B protein was purified by affinity chromatography, and crosslinking assays were performed to determine the presence of oligomers. Hemolysis assays and transmission electron microscopy were performed to identify structures involved in permeability changes. Results The DENV-2 NS2B protein showed similitude with the JEV viroporin. The DENV-2 NS2B protein possessed the ability to change the membrane permeability in bacteria, to restrict bacterial cell growth, and to enable membrane permeability to hygromycin B. The NS2B protein formed trimers that could participate in cell lysis and generate organized structures on eukaryotes membranes. Conclusions Our data suggest that the DENV-2 NS2B viral protein is capable of oligomerizing and organizing to form pore-like structures in different lipid environments, thereby modifying the permeability of cell membranes.