Intracellular Sequestration of the NKG2D Ligand MIC B by Species F Adenovirus.

The enteric human adenoviruses of species F (HAdVs-F), which comprise HAdV-F40 and HAdV-F41, are significant pathogens that cause acute gastroenteritis in children worldwide. The early transcription unit 3 (E3) of HAdVs-F is markedly different from that of all other HAdV species. To date, the E3 proteins unique to HAdVs-F have not been characterized and the mechanism by which HAdVs-F evade immune defenses in the gastrointestinal (GI) tract is poorly understood. Here, we show that HAdV-F41 infection of human intestinal HCT116 cells upregulated the expression of MHC class I-related chain A (MIC A) and MIC B relative to uninfected cells. Our results also showed that, for MIC B, this response did not however result in a significant increase of MIC B on the cell surface. Instead, MIC B was largely sequestered intracellularly. Thus, although HAdV-F41 infection of HCT116 cells upregulated MIC B expression, the ligand remained inside infected cells. A similar observation could not be made for MIC A in these cells. Our preliminary findings represent a novel function of HAdVs-F that may enable these viruses to evade immune surveillance by natural killer (NK) cells in the infected gut, thereby paving the way for the future investigation of their unique E3 proteins.

Using machine learning to develop a novel COVID-19 Vulnerability Index (C19VI).

COVID-19 is now one of the most leading causes of death in the United States (US). Systemic health, social and economic disparities have put the minorities and economically poor communities at a higher risk than others. There is an immediate requirement to develop a reliable measure of county-level vulnerabilities that can capture the heterogeneity of vulnerable communities. This study reports a COVID-19 Vulnerability Index (C19VI) for identifying and mapping vulnerable counties. We proposed a Random Forest machine learning-based vulnerability model using CDC's sociodemographic and COVID-19-specific themes. An innovative 'COVID-19 Impact Assessment' algorithm was also developed for evaluating severity of the pandemic and to train the vulnerability model. Developed C19VI was statistically validated and compared with the CDC COVID-19 Community Vulnerability Index (CCVI). Finally, using C19VI and the census data, we explored racial inequalities and economic disparities in COVID-19 health outcomes. Our index indicates that 575 counties (45 million people) fall into the 'very high' vulnerability class, 765 counties (66 million people) in the 'high' vulnerability class, and 1435 counties (204 million people) in the 'moderate' or 'low' vulnerability class. Only 367 counties (20 million people) were found as 'very low' vulnerable areas. Furthermore, C19VI reveals that 524 counties with a racial minority population higher than 13% and 420 counties with poverty higher than 20% are in the 'very high' or 'high' vulnerability classes. The C19VI aims at helping public health officials and disaster management agencies to develop effective mitigation strategies especially for the disproportionately impacted communities.

Fatal Dengue Cases Reveal Brain Injury and Viral Replication in Brain-Resident Cells Associated with the Local Production of Pro-Inflammatory Mediators.

Dengue is an arboviral disease caused by dengue virus (DENV), which is transmitted to humans by Aedes aegypti mosquitoes. Infection by DENV most commonly results in a mild flu-like illness; however, the disease has been increasingly associated with neurological symptomatology. This association draws attention to further investigations on the impact of DENV infection in the host's central nervous system. Here, we analyzed brain samples of three fatal dengue cases that occurred in 2002 during an outbreak in Rio de Janeiro, Brazil. Brain tissues of these cases were marked by histopathological alterations, such as degenerated neurons, demyelination, hemorrhage, edema, and increased numbers of astrocytes and microglial cells. Samples were also characterized by lymphocytic infiltrates mainly composed of CD8 T cells. DENV replication was evidenced in neurons, microglia and endothelial cells through immunohistochemistry and in situ hybridization techniques. Pro-inflammatory cytokines, such as TNF-α and IFN-γ were detected in microglia, while endothelial cells were marked by the expression of RANTES/CCL5. Cytoplasmic HMGB1 and the production of nitric oxide were also found in neurons and microglial cells. This work highlights the possible participation of several local pro-inflammatory mediators in the establishment of dengue neuropathogenesis.

Immune evasion by adenoviruses: a window into host-virus adaptation.

Human adenoviruses (HAdVs) are widespread pathogens that cause a number of partially overlapping, species-specific infections associated with respiratory, urinary, gastrointestinal, and ocular diseases. The early 3 (E3) region of adenoviruses is highly divergent between different species, and it encodes a multitude of proteins with immunomodulatory functions. The study of genetic diversity in the E3 region offers a unique opportunity to gain insight into how the various HAdVs have evolutionarily adapted in response to the selection pressures exerted by host immune defenses. The objective of this review was to discuss subversion of host antiviral immune responses by HAdVs, with a focus on suppression of MHC class I antigen presentation, as a window into host-HAdV adaptation.

mTORC1-mediated polarization of M1 macrophages and their accumulation in the liver correlate with immunopathology in fatal ehrlichiosis.

A polarized macrophage response into inflammatory (M1) or regenerative/anti-inflammatory (M2) phenotypes is critical in host response to multiple intracellular bacterial infections. Ehrlichia is an obligate Gram-negative intracellular bacterium that causes human monocytic ehrlichiosis (HME): a febrile illness that may progress to fatal sepsis with multi-organ failure. We have shown that liver injury and Ehrlichia-induced sepsis occur due to dysregulated inflammation. Here, we investigated the contribution of macrophages to Ehrlichia-induced sepsis using murine models of mild and fatal ehrlichiosis. Lethally-infected mice showed accumulation of M1 macrophages (iNOS-positive) in the liver. In contrast, non-lethally infected mice showed polarization of M2 macrophages and their accumulation in peritoneum, but not in the liver. Predominance of M1 macrophages in lethally-infected mice was associated with expansion of IL-17-producing T, NK, and NKT cells. Consistent with the in vivo data, infection of bone marrow-derived macrophages (BMM) with lethal Ehrlichia polarized M0 macrophages into M1 phenotype under an mTORC1-dependent manner, while infection with non-lethal Ehrlichia polarized these cells into M2 types. This work highlights that mTORC1-mediated polarization of macrophages towards M1 phenotype may contribute to induction of pathogenic immune responses during fatal ehrlichiosis. Targeting mTORC1 pathway may provide a novel aproach for treatment of HME.

Emerging Roles of Autophagy and Inflammasome in Ehrlichiosis.

Human monocytic ehrlichiosis (HME) is a potentially life-threatening tick-borne rickettsial disease (TBRD) caused by the obligate intracellular Gram-negative bacteria, Ehrlichia. Fatal HME presents with acute ailments of sepsis and toxic shock-like symptoms that can evolve to multi-organ failure and death. Early clinical and laboratory diagnosis of HME are problematic due to non-specific flu-like symptoms and limitations in the current diagnostic testing. Several studies in murine models showed that cell-mediated immunity acts as a "double-edged sword" in fatal ehrlichiosis. Protective components are mainly formed by CD4 Th1 and NKT cells, in contrast to deleterious effects originated from neutrophils and TNF-α-producing CD8 T cells. Recent research has highlighted the central role of the inflammasome and autophagy as part of innate immune responses also leading to protective or pathogenic scenarios. Recognition of pathogen-associated molecular patterns (PAMPS) or damage-associated molecular patterns (DAMPS) triggers the assembly of the inflammasome complex that leads to multiple outcomes. Recognition of PAMPs or DAMPs by such complexes can result in activation of caspase-1 and -11, secretion of the pro-inflammatory cytokines IL-1ß and IL-18 culminating into dysregulated inflammation, and inflammatory cell death known as pyroptosis. The precise functions of inflammasomes and autophagy remain unexplored in infections with obligate intracellular rickettsial pathogens, such as Ehrlichia. In this review, we discuss the intracellular innate immune surveillance in ehrlichiosis involving the regulation of inflammasome and autophagy, and how this response influences the innate and adaptive immune responses against Ehrlichia. Understanding such mechanisms would pave the way in research for novel diagnostic, preventative and therapeutic approaches against Ehrlichia and other rickettsial diseases.

Placental Inflammation and Fetal Injury in a Rare Zika Case Associated With Guillain-Barré Syndrome and Abortion.

Zika virus (ZIKV) is an emerging virus involved in recent outbreaks in Brazil. The association between the virus and Guillain-Barré syndrome (GBS) or congenital disorders has raised a worldwide concern. In this work, we investigated a rare Zika case, which was associated with GBS and spontaneous retained abortion. Using specific anti-ZIKV staining, the virus was identified in placenta (mainly in Hofbauer cells) and in several fetal tissues, such as brain, lungs, kidneys, skin and liver. Histological analyses of the placenta and fetal organs revealed different types of tissue abnormalities, which included inflammation, hemorrhage, edema and necrosis in placenta, as well as tissue disorganization in the fetus. Increased cellularity (Hofbauer cells and TCD8+ lymphocytes), expression of local pro-inflammatory cytokines such as IFN-γ and TNF-α, and other markers, such as RANTES/CCL5 and VEGFR2, supported placental inflammation and dysfunction. The commitment of the maternal-fetal link in association with fetal damage gave rise to a discussion regarding the influence of the maternal immunity toward the fetal development. Findings presented in this work may help understanding the ZIKV immunopathogenesis under the rare contexts of spontaneous abortions in association with GBS.

BALB/c mice infected with DENV-2 strain 66985 by the intravenous route display injury in the central nervous system.

Dengue is a mild flu-like arboviral illness caused by dengue virus (DENV) that occurs in tropical and subtropical countries. An increasing number of reports have been indicating that dengue is also associated to neurological manifestations, however, little is known regarding the neuropathogenesis of the disease. Here, using BALB/c mice intravenously infected with DENV-2 strain 66985, we demonstrated that the virus is capable of invading and damaging the host's central nervous system (CNS). Brain and cerebellum of infected animals revealed histological alterations such as the presence of inflammatory infiltrates, thickening of pia matter and disorganization of white matter. Additionally, it was also seen that infection lead to altered morphology of neuroglial cells and apoptotic cell death. Such observations highlighted possible alterations that DENV may promote in the host's CNS during a natural infection, hence, helping us to better understand the neuropathological component of the disease.

Correction: Peripheral Organs of Dengue Fatal Cases Present Strong Pro-Inflammatory Response with Participation of IFN-Gamma-, TNF-Alpha- and RANTES-Producing Cells.

[This corrects the article DOI: 10.1371/journal.pone.0168973.].

Dengue fatal cases present virus-specific HMGB1 response in peripheral organs.

Dengue is an important infectious disease that presents high incidence and yields a relevant number of fatal cases (about 20,000) every year worldwide. Despite its epidemiological relevance, there are many knowledge gaps concerning dengue pathogenesis, especially with regards to the circumstances that drive a mild clinical course to a severe disease. In this work, we investigated the participation of high mobility group box 1 (HMGB1), an important modulator of inflammation, in dengue fatal cases. Histopathological and ultrastructural analyses revealed that liver, lung and heart post-mortem samples were marked by tissue abnormalities, such as necrosis and apoptotic cell death. These observations go in line with an HMGB1-mediated response and raised concerns regarding the participation of this cytokine in promoting/perpetuating inflammation in severe dengue. Further experiments of immunohistochemistry (IHC) showed increased expression of cytoplasmic HMGB1 in dengue-extracted tissues when compared to non-dengue controls. Co-staining of DENV RNA and HMGB1 in the host cell cytoplasm, as found by in situ hybridization and IHC, confirmed the virus specific induction of the HMGB1-mediated response in these peripheral tissues. This report brings the first in-situ evidence of the participation of HMGB1 in severe dengue and highlights novel considerations in the development of dengue immunopathogenesis.

New insights into flavivirus biology: the influence of pH over interactions between prM and E proteins.

Diseases caused by flaviviruses, such as dengue and zika, are globally recognized as major threats. During infection, a critical point in their replicative cycle is the maturation step, which occurs throughout the cellular exocytic pathway. This step is a pH-dependent process that involves the modification of the viral envelope by converting prM (pre-membrane) into M (membrane) proteins with the release of a "pr peptide". After this reaction, the pr peptides remain bound to the viral envelope while the virions cross the acidic trans-Golgi network, and are released only at neutral pH after secretion of the virus particles. Despite this current knowledge, the molecular basis of the flavivirus maturation step is largely unknown. Here, based on the crystal structure of the dengue pr-E complex ("pr peptide" bound to virus envelope protein) and using molecular dynamics simulations, we found that the pH shift from acidic to neutral yields considerable structural changes in the system. Dynamic cross correlation maps and root mean square deviation analyses revealed that the pr-E junction is clearly unstable under neutral pH. Secondary structure analysis also revealed that the fusion loop region, present in the E protein, is sensitive to pH and tends to unstructure at a neutral environment. Moreover, we found that five residues present in the E protein, Gly102, His244, Thr70, Thr68 and Asn67 are critical to confer stability to the pr-E complex while inside the Golgi apparatus. This work brings details about the dynamical behavior of the pr-E system, helps to better understand the flavivirus biology and may also be of use in the development of novel antiviral strategies.

Insights into CC Chemokine Ligand 2/Chemokine Receptor 2 Molecular Recognition: A Step Forward toward Antichemotactic Agents.

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is a chemokine that recruits immune cells to inflammatory sites by interacting with G protein-coupled receptor CCR2. The CCL2/CCR2 axis is also involved in pathological processes such as tumor growth and metastasis and hence is currently considered as an important drug target. CCL2 exists in a dynamic monomer-dimer equilibrium that is modulated by CCR2 binding. We used solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to study the interactions between CCL2 and a sulfopeptide corresponding to the N-terminal sequence of CCR2 (CCR218-31). Peptide binding induced the dissociation of CCL2 into monomers, forming stable CCL2/CCR218-31 complexes. NMR relaxation measurements indicated that residues around the CCR218-31 binding site, which are located at the dimer interface, undergo a complex regime of motions. NMR data were used to construct a three-dimensional structural model of the CCL2/CCR218-31 complex, revealing that CCR218-31 occupies a binding site juxtaposed to the dimer interface, partially replacing monomer-monomer contacts, explaining why CCR218-31 binding weakens the dimer interface and induces dissociation. We found that the main interactions governing receptor binding are highly stable salt bridges with conserved chemokine residues as well as hydrophobic interactions. These data provide new insights into the structure-function relationship of the CCL2-CCR2 interaction and may be helpful for the design of novel antichemotactic agents.

The flavivirus capsid protein: Structure, function and perspectives towards drug design.

Flaviviruses, such as dengue and zika viruses, are etiologic agents transmitted to humans mainly by arthropods and are of great epidemiological interest. The flavivirus capsid protein is a structural element required for the viral nucleocapsid assembly that presents the classical function of sheltering the viral genome. After decades of research, many reports have shown its different functionalities and influence over cell normal functioning. The subcellular distribution of this protein, which involves accumulation around lipid droplets and nuclear localization, also corroborates with its multi-functional characteristic. As flavivirus diseases are still in need of global control and in view of the possible key functionalities that the capsid protein promotes over flavivirus biology, novel considerations arise towards anti-flavivirus drug research. This review covers the main aspects concerning structural and functional features of the flavivirus C protein, ultimately, highlighting prospects in drug discovery based on this viral target.

Peripheral Organs of Dengue Fatal Cases Present Strong Pro-Inflammatory Response with Participation of IFN-Gamma-, TNF-Alpha- and RANTES-Producing Cells.

Dengue disease is an acute viral illness caused by dengue virus (DENV) that can progress to hemorrhagic stages leading to about 20000 deaths every year worldwide. Despite many clinical investigations regarding dengue, the immunopathogenic process by which infected patients evolve to the severe forms is not fully understood. Apart from differences in virulence and the antibody cross reactivity that can potentially augment virus replication, imbalanced cellular immunity is also seen as a major concern in the establishment of severe dengue. In this context, the investigation of cellular immunity and its products in dengue fatal cases may provide valuable data to help revealing dengue immunopathogenesis. Here, based in four dengue fatal cases infected by the serotype 3 in Brazil, different peripheral organs (livers, lungs and kidneys) were studied to evaluate the presence of cell infiltrates and the patterns of local cytokine response. The overall scenario of the studied cases revealed a considerable systemic involvement of infection with mononuclear cells targeted to all of the evaluated organs, as measured by immunohistochemistry (IHC). Quantification of cytokine-expressing cells in peripheral tissues was also performed to characterize the ongoing inflammatory process by the severe stage of the disease. Increased levels of IFN-γ- and TNF-α-expressing cells in liver, lung and kidney samples of post-mortem subjects evidenced a strong pro-inflammatory induction in these tissues. The presence of increased RANTES-producing cell numbers in all analyzed organs suggested a possible link between the clinical status and altered vascular permeability. Co-staining of DENV RNA and IFN-γ or TNF-α using in situ hibridization and IHC confirmed the virus-specific trigger of the pro-inflammatory response. Taken together, this work provided additional evidences that corroborated with the traditional theories regarding the "cytokine storm" and the occurrence of uneven cellular immunity in response to DENV as major reasons for progress to severe disease.

Aspects of T Cell-Mediated Immunity Induced in Mice by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the Intracerebral Route.

Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy.

The mechanism by which P250L mutation impairs flavivirus-NS1 dimerization: an investigation based on molecular dynamics simulations.

The flavivirus non-structural protein 1 (NS1) is a conserved glycoprotein with as yet undefined biological function. This protein dimerizes when inside infected cells or associated to cell membranes but also forms lipid-associated hexamers when secreted to the extracellular space. A single amino acid substitution (P250L) is capable of preventing the dimerization of NS1 resulting in lower virulence and slower virus replication. In this work, based on molecular dynamics simulations of the dengue-2 virus NS1 [Formula: see text]-ladder monomer as a core model, we found that this mutation can induce several conformational changes that importantly affect critical monomer-monomer interactions. Based on additional simulations, we suggest a mechanism by which a highly orchestrated sequence of events propagate the local perturbations around the mutation site towards the dimer interface. The elucidation of such a mechanism could potentially support new strategies for rational production of live-attenuated vaccines and highlights a step forward in the development of novel anti-flavivirus measures.

Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen.

Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD50), which was abrogated with the increase of viral dose (40 LD50). The pcTPANS1 also induced activation of CD4+ and CD8+ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8+ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4+ cells. Depletion of CD4+ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4+ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4+ T cells and the humoral immunity.

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination.

Potency determination of recombinant IFN-alpha based on phosphorylated STAT1 using flow cytometry.

The interferon (IFN) family of cytokines is recognized as a key component of the innate immune response and the first line of defense against viral infection. The usage of the IFN-alpha as a biopharmaceutical has been mainly applied in the treatment of chronic hepatitis C. In the literature it is possible to find a great variety of methods to determine the potency of these cytokines, and many efforts have been made in order to develop practical bioassays to study the biological activity of IFNs. In this technical note, we present a different approach to determine the potency of a recombinant IFN-alpha preparation based on the activation of the signal transducers and activators of transcription 1 (STAT1) using flow cytometry technique. Under the conditions of this study, this new approach proved to be useful and promising to assess the potency of these biopharmaceuticals and may also be used as an important tool in the quality control of such biological products.