A host-dependent transcriptional response correlates with Dengue virus infection rate in healthy humans.
Ontology highlight
ABSTRACT: Dengue virus (DENV) infection is a major emerging disease in tropical and subtropical countries and the influence of host genetics during early phases of infection remains to be fully elucidated. Here we use dendritic cells (DCs) and macrophages from healthy individuals to establish inter-individual variability in DENV infection and antibody-mediated enhancement (ADE). We show that host-related factors determine the severity of the infection rate both in DENV-infected DCs and macrophages following ADE. We then correlate the inter-individual variability in infection rates with genome-wide transcript abundance measured by RNA sequencing in DCs following DENV infection. We report 190 host-related transcripts in DCs that correlate markedly with infection rates that form an ubiquitin-mediated antiviral network. Furthermore, these transcripts are enriched for glycolysis, which we show is critical for the early phases of infection. Among virus induced transcripts (i.e. significant correlation only after DENV), we identify DUSP10 as the best indicator of the severity of infection. These results indicate the importance of host genetics in shaping the severity of DENV infection rates in DCs and identify novel potential DENV-susceptibility targets.
Project description:Dengue virus (DENV) can hijack non-neutralizing IgG antibodies to facilitate its uptake into target cells expressing Fc gamma receptors (FcgR) - a process known as antibody-dependent enhancement (ADE) of infection. Beyond a requirement for FcgR, host dependency factors for this non-canonical infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout screens in an in vitro system permissive to infection only in the presence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates binding and internalization of IgG-bound DENV but is not required for canonical infection. Additionally, we identified host factors with no previously described role in DENV infection, including TBC1D24 and SV2B, both of which have known functions in regulated secretion. Using genetic knockout and trans-complemented cells, we validated a functional requirement for these host factors in ADE assays performed with monoclonal antibodies and polyclonal sera in multiple cell lines and using all four DENV serotypes. We show that TBC1D24 and SV2B promote binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that are required for ADE of DENV infection. Our findings represent a first step towards advancing fundamental knowledge behind the biology of ADE that can ultimately be exploited to inform vaccination and therapeutic approaches.
Project description:Dengue virus (DENV) is the causative agent of dengue, a mosquito-borne disease that represents a significant and growing public health burden around the world. A unique pathophysiological feature of dengue is immune-mediated enhancement, wherein preexisting immunity elicited by a primary infection can enhance the severity of a subsequent infection by a heterologous DENV serotype. A leading mechanistic explanation for this phenomenon is antibody dependent enhancement (ADE), where sub-neutralizing concentrations of DENV-specific IgG antibodies facilitate entry of DENV into FcR expressing cells such as monocytes, macrophages, and dendritic cells. Accordingly, this model posits that phagocytic mononuclear cells are the primary reservoir of DENV. However, analysis of samples from individuals experiencing acute DENV infection reveals that B cells are the largest reservoir of infected circulating cells, representing a disconnect in our understanding of immune-mediated DENV tropism. In this study, we demonstrate that the expression of a DENV-specific B cell receptor (BCR) renders cells highly susceptible to DENV infection, with the infection-enhancing activity of the membrane-restricted BCR correlating with the ADE potential of the IgG version of the antibody. In addition, we observed that the frequency of DENV-infectable B cells increases in previously flavivirus-naïve volunteers after a primary DENV infection. These findings suggest that BCR-dependent infection of B cells is a novel mechanism immune-mediated enhancement of DENV-infection.
Project description:Background: Dengue virus (DENV) infection often leads to acute illness lasting 2-7 days with severity ranging from dengue fever (DF) to hemorrhagic fever (DHF) and fatal dengue shock syndrome (DSS). The dynamic changes of host responses on the gene transcription level that accompany DENV infection and differences between DF and DHF cases have been poorly understood, particularly for South American population. Methodology/Principal Findings: Supported by a longitudinal active surveillance program for dengue transmission in Maracay, Venezuela, we conducted a prospective study to investigate host responses in dengue patients. Blood specimens and clinical information were collected on a daily basis from febrile cases confirmed with DENV infection from their first day of enrollment to early defervescence together with one convalescent sample. A total of 49 and 13 study participants were defined as DF and DHF cases respectively based on their clinical and hematological information. Using convalescent specimens as baseline, day-to-day gene expression was evaluated ex vivo in peripheral blood mononuclear cells of the study participants. Two waves of gene expression were detected: the first wave peaked at day 1 from the onset of fever (day 0) then declined at days 3-4; the second wave emerged from day 4 and peaked around day 5-6. Genes associated with innate immune process, including type I interferon signaling, cytokine-mediated signaling, chemotaxis, and antiviral responses, dominated the first wave; whereas genes involved in cell cycle processes, including cell division, mitosis, DNA replication, chromosome, and spindle organization dominated the second wave. Measureable genomic markers predicted early acute, late acute and convalescent phases with 91% accuracy. Gene signatures expressed in early acute phase predicted disease severity (DF vs DHF) with 96% accuracy. Conclusions/Significance: Our study established a dynamic pattern of detailed host immune responses to DENV infection and revealed genomic signatures valuable for diagnostics purposes.
Project description:Background: Dengue virus (DENV) infection often leads to acute illness lasting 2-7 days with severity ranging from dengue fever (DF) to hemorrhagic fever (DHF) and fatal dengue shock syndrome (DSS). The dynamic changes of host responses on the gene transcription level that accompany DENV infection and differences between DF and DHF cases have been poorly understood, particularly for South American population. Methodology/Principal Findings: Supported by a longitudinal active surveillance program for dengue transmission in Maracay, Venezuela, we conducted a prospective study to investigate host responses in dengue patients. Blood specimens and clinical information were collected on a daily basis from febrile cases confirmed with DENV infection from their first day of enrollment to early defervescence together with one convalescent sample. A total of 49 and 13 study participants were defined as DF and DHF cases respectively based on their clinical and hematological information. Using convalescent specimens as baseline, day-to-day gene expression was evaluated ex vivo in peripheral blood mononuclear cells of the study participants. Two waves of gene expression were detected: the first wave peaked at day 1 from the onset of fever (day 0) then declined at days 3-4; the second wave emerged from day 4 and peaked around day 5-6. Genes associated with innate immune process, including type I interferon signaling, cytokine-mediated signaling, chemotaxis, and antiviral responses, dominated the first wave; whereas genes involved in cell cycle processes, including cell division, mitosis, DNA replication, chromosome, and spindle organization dominated the second wave. Measureable genomic markers predicted early acute, late acute and convalescent phases with 91% accuracy. Gene signatures expressed in early acute phase predicted disease severity (DF vs DHF) with 96% accuracy. Conclusions/Significance: Our study established a dynamic pattern of detailed host immune responses to DENV infection and revealed genomic signatures valuable for diagnostics purposes. Total 64 subjects representing 51 DF (dengue fever) and 13 DHF (dengue hemorrhagic fever) cases were used to study gene expression during the course of dengue acute illness. Sample information regarding Patient No., Infecting serotype, fever status (fever days or defervescent (df) days), and disease severity (DF vs DHF) were provided. The date for a sample for gene chip hybridization and image scanning was marked as Scan date. Samples from study subjects were collected once a patient was enrolled. Over 200 samples were collected. Two genechip platforms were used, the HG-focus and HG-U133plus2. A total of 168 samples were assayed on the HG-focus platform, 2 were excluded for further analysis as they failed on quality control (i.e. VFP-0003_G7_DF, VFP-0029_G1_DF samples). A total of 101 samples were assayed on the HG-U133plus2 platform, 4 were excluded for further analysis as they failed on quality control (i.e. VFP_0186_G4_DF, VFP-0213_G3_DF, VFP_0213_G5_DF, VFP_0220_G3_DHF). Data generated by HG-focus platform were used to explore significantly expressed genes in individual Gs, phases, and for prediction analysis for disease phases. Whereas data generated by HG-U133plus2 were first used to assess the reproducibility of results generated by the HG-focus platform. Secondarily, they were used to perform prediction analysis for disease severity. Samples were grouped into stages (G) based on the timepoint when they were collected: G0=day0 (on the day of fever onset); G1=day 1 (one day after fever onset); G2=day2 (two days after fever onset)M-bM-^@M-&until G5; G6=day6-10 (6-10 days after fever onset); G7= convalescent time point (around day 28 after the first sample). Samples were also grouped into early acute (G0-G3), late acute (G4-G6) and convalescent phases (G7). References for the protocols; PMID 12803996 and 20078211
Project description:Background: Dengue virus (DENV) infection has a global impact on public health. The clinical outcomes (of DENV) can vary from a flu-like illness called dengue fever (DF), to a more severe form, known as dengue hemorrhagic fever (DHF). The underlying innate immune mechanisms leading to protective or detrimental outcomes have not been fully elucidated. Helper innate lymphoid cells (hILCs), an innate lymphocyte recently discovered, functionally resemble T-helper cells and are important in inflammation and homeostasis. However, the role of hILCs in DENV infection had been unexplored. Methods: We performed flow cytometry to investigate the frequency and phenotype of hILCs in peripheral blood mononuclear cells from DENV-infected patients of different disease severities (DF and DHF), and at different phases (febrile and convalescence) of infection. Intracellular cytokine staining of hILCs from DF and DHF were also evaluated by flow cytometry after ex vivo stimulation. Further, the hILCs were sorted and subjected to transcriptome analysis using RNA sequencing. Differential gene expression analysis was performed to compare the febrile and convalescent phase samples in DF and DHF. Selected differentially expressed genes were then validated by quantitative PCR. Results: Phenotypic analysis showed marked activation of all three hILC subsets during the febrile phase as shown by higher CD69 expression when compared to paired convalescent samples, although the frequency of hILCs remained unchanged. Upon ex vivo stimulation, hILCs from febrile phase DHF produced significantly higher IFN-g and IL-4 when compared to those of DF. Transcriptomic analysis showed unique hILCs gene expression in DF and DHF, suggesting that divergent functions of hILCs may be associated with different disease severities. Differential gene expression analysis indicated that hILCs function both in cytokine secretion and cytotoxicity during the febrile phase of DENV infection. Conclusions: Helper ILCs are activated in the febrile phase of DENV infection and display unique transcriptomic changes as well as cytokine production that correlate with severity. Targeting hILCs during early innate response to DENV might help shape subsequent immune responses and potentially lessen the disease severity in the future.
Project description:The ability of many viruses to manipulate the host antiviral immune response often results in complex host-pathogen interactions. In order to study the interaction of dengue virus (DENV) with the Aedes aegypti immune response, we have characterized the DENV infection-responsive transcriptome of the immune-competent A. aegypti cell line Aag2. As in mosquitoes, DENV infection transcriptionally activated the cell line Toll pathway and a variety of cellular physiological systems. Most notably, however, DENV infection down-regulated the expression levels of numerous immune signaling molecules and antimicrobial peptides (AMPs). Functional assays showed that transcriptional induction of AMPs from the Toll and IMD pathways in response to bacterial challenge is impaired in DENV-infected cells. In addition, Escherichia coli, a gram-negative bacteria species, grew better when co-cultured with DENV-infected cells than with uninfected cells, suggesting a decreased production of AMPs from the IMD pathway in virus-infected cells. Pre-stimulation of the cell line with gram-positive bacteria prior to DENV infection had no effect on DENV titers, while pre-stimulation with gram-negative bacteria resulted in an increase in DENV titers. These results indicate that DENV is capable of actively suppressing immune responses in the cells it infects, a phenomenon that may have important consequences for virus transmission and insect physiology. Infected (dengue virus or heat-inactivated dengue virus) vs. naive cells. 3 replicates each.
Project description:Controlled dengue human challenge studies present a unique opportunity to address many longstanding questions in the field of flavivirus biology. These fundamental questions include defining the early immunological signatures of infection, the host/environmental factors that impact disease severity, and the role of preexisting immunity on the development of symptomatic viral infection. However, while several controlled dengue human challenge studies have been performed and appear to clinically recapitulate may features of mild natural DENV infection, limited data are available on how the immunological and transcriptional response elicited by these attenuated challenge viruses compares to the profile associated with a natural, unattenuated DENV infection. To bridge this knowledge gap, we performed scRNAseq analysis on longitudinally collected PBMC samples obtained from 3 individuals (8 time points per subject) enrolled in the SUNY/WRAIR DENV-1 controlled human challenge study. In addition, 3 time points (two acute infection time points, one control time point) from two individuals experiencing a natural DENV-1 infection were analyzed and computationally integrated with the challenge model dataset. This temporally integrated dataset contains a total of 171,208 cells and 22 statistically distinct populations corresponding to all major anticipated leukocyte subsets. While all identified cell populations demonstrated significant and consistant perturbations in their transcriptional profile in response to either natural or experimental DENV infection, conventional monocytes respond most robustly to infection across all subjects and study groups from an unbiased transcriptional perspective. Using these data, core sets of genes that were consistently induced by either natural or experimental DENV were identified, and the overlap between the two arms of the study assessed.
Project description:Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we reported on the first genome-wide host transcriptional responses to normal PRRSV (N-PRRSV) infection using Solexa/Illumina’s digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyzed systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results indicated that N-PRRSV appeared to utilize multiple strategies for its long surviral in infected pigs, including subverting host innate immune response, hijacking host lipid metabolism, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes production and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processing. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection or developed secondary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS.