Following infection, the virus initially replicates in skin cells, such as keratinocytes and Langerhans cells This will trigger a variety of host innate immune responses.
Innate immune cells are the first to respond to infection by using pattern recognition receptor PRR recognizing pathogen-associated molecular patterns 30 , These immune cells include DCs, macrophages, and monocytes. PRR recognition will trigger production of cytokines and chemokines, which induce an antiviral state.
Thus, they are an essential part of the innate immune response against virus, sensing viral replication in the cytoplasm. Double membrane vacuoles, called autophagosomes, will engulf foreign cytoplasmic material and fuse with the lysosome for degradation, inhibiting virus replication red. Cleavage of C4 and C2 by MBL-associated serine protease-2 MASP-2 make the C3 convertase and initiates the classical complement cascade, including the formation of C5 convertase and the C5b-9 membrane attack complex MAC to induce lysis, recruitment of phagocytes, and inflammation.
The complement system is also an important part of innate immune response to the virus. The following classical complement cascade includes the formation of C5 convertase and the C5b-9 membrane attack complex MAC to induce lysis, recruitment of phagocytes, and inflammation 45 Fig.
There is also evidence of vertebrate systems using the RNAi to inhibit virus infection, though it is not as well understood. Autophagy is a natural cellular process to maintain homeostasis, regulating cell degradation usually in response to starvation 50 and disease Double membrane vacuoles, called autophagosomes, will engulf cytoplasmic material and fuse with the lysosome for degradation.
Thus, autophagy is an important system to clearing the host of foreign pathogens such as viruses. Autophagy has been shown to be activated in DENV infections and has antiviral or pro-viral activity depending on cell type. Autophagy inhibits replication in monocytes, specifically under ADE conditions, which make these cells highly susceptible to infection In liver cells, autophagy has a pro-viral effect.
DENV blocks the autophagosome from fusing with the lysosome, and instead uses the vacuoles for replication 54 , assembly and maturation 55 , and evading neutralizing antibodies during transmission Apoptosis is a highly regulated process of self-destruction that cells undergo in response to stimuli such as redundant or dangerous cells like tumors or pathogen-infected cells. There are two main apoptotic pathways, the intrinsic or mitochondrial and extrinsic, though the two are linked and converge at the execution phase, where the cell undergoes DNA fragmentation, degradation of the cytoskeleton, and formation of apoptotic bodies that are ultimately engulfed by surrounding phaocytes DENV proteins have been shown to activate apoptosis inside infected cells.
The capsid protein nuclear localization interacts with death-associated protein 6 and triggers Fas-mediated apoptosis in liver cells The intrinsic pathway is activated by the DENV membrane protein ectodomain export from golgi to plasma membrane Some immune responses are implicated with disease severity. TLR4 recognition of NS1 leads to pro-inflammatory cytokine production that contribute to vascular damage 63 Fig.
NS1 will also exacerbate disease by binding to uninfected cells to initiate vascular leakage 64 , Activation of the alternative complement pathway is associated with disease severity It is unclear if increased number of NK cells contribute to increase in disease severity.
However, acutely infected patients in Thailand showed no correlation between NK cell subsets and level of severity It also can block pathways that alert nearby cells of infection. Nonstructural proteins block signaling pathways after virus recognition and inhibit type I IFN production. DENV will exploit the autophagy pathway and use autophagosomes for replication, assembly and maturation, and evasion of neutralizing antibodies during transmission red. DENV will subvert apoptosis early in the life cycle to ensure viral replication.
The virus evades the complement response using NS1. Other DENV proteins have been shown to have immune evasion activity.
DENV C protein aids in inhibition of apoptosis in Huh7 cells by interacting with calcium-modulating cyclophilin-binding ligand, an ER protein associated with cell survival by regulating Bim-dependent death There is currently one licensed vaccine, Dengvaxia by Sanofi-Pasteur, which uses the prM-E dengue sequence in a yellow fever virus backbone. There are no approved antivirals for DENV. Treatment for clinical manifestations of the acute febrile are paracetamol for high fever and oral or intravenous fluid intake Recent DENV antiviral research has been focusing on identifying novel compounds that targeting the DENV proteins responsible for replication and innate immune evasion.
The novel small-molecule compound, ST, inhibits DENV replication by targeting the capsid protein and reduced viremia and viral load in mice Additionally, a therapeutic monoclonal antibody raised against NS1 induced complement-mediated lysis in vitro and had protective effects in vivo Drug repurposing have been investigated for DENV inhibition.
Minocycline, typically used as an antibiotic and anti-inflammatory, inhibits DENV replication in vitro by suppressing migration inhibitory facto, a catalyst for autophagy, and this drug also decreased viremia and autophagy formation in vivo Secreted phospholipase A 2 , obtained from snake venom showed viricidal, has neutralizing activity against DENV, targeting the virus envelope lipid bilayer DENV is a mosquito-borne Flavivirus that is endemic in many tropical and sub-tropical countries.
The NS proteins are responsible for viral replication and host innate immune evasion. The innate immune response to DENV is not well characterized nor are the exact roles of the NS proteins in evading the immune response. Other innate immune responses include complement activation, apoptosis, autophagy, and RNAi.
It is important to understand the host innate immune response to infection and how the virus evades or exploits this in order to develop effective antivirals and vaccines. In addition, T. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. National Center for Biotechnology Information , U.
Journal List Emerg Microbes Infect v. Emerg Microbes Infect. Published online Oct Naoko Uno 1 and Ted M. Ross 1, 2. Ted M. Author information Article notes Copyright and License information Disclaimer. Ross, Email: ude. Corresponding author. This article has been cited by other articles in PMC. Abstract Dengue virus DENV is a mosquito-borne Flavivirus that is endemic in many tropical and sub-tropical countries where the transmission vectors Aedes spp.
Dengue virus Epidemiology Dengue virus DENV is the most prevalent arbovirus worldwide, found in over tropical and sub-tropical countries 1. Open in a separate window. The viral life cycle of dengue virus DENV. Viral life cycle DENV is spread to humans from an infected mosquito. Innate immune response to DENV infection.
Complement system response The complement system is also an important part of innate immune response to the virus. Innate immunity associated with severe disease Some immune responses are implicated with disease severity. DENV evasion of innate immune response. We observed a reduction in ROS in infected and differentiating cells through an apparent activation of NFE2L2 transcription factor activity.
Further, this reduction in ROS promoted virus replication while simultaneously interfering with optimum differentiation of the megakaryocyte mother cell. All cells were regularly checked for visible bacterial and fungal contamination or Mycoplasma contamination by PCR-based detection from cell-free culture supernatant.
Fresh dilutions were used for every experiment. Vero cell monolayers in well plate were infected with fold serial dilutions of virus inoculum. After incubation with primary antibody, the cells were washed twice with PBS before addition of secondary antibody dilutions. The permeabilized cells were sequentially stained with dilution of 4G2 purified IgG2a mAb produced in the lab from Hybridoma-HB antibody and anti-mouse Alexa conjugated secondary antibody.
Subsequently, the cells were washed with PBS and analyzed by flow cytometry. K cells were washed with PBS, fixed and permeabilized as described earlier for immunostaining purposes. Cufflinks 2. For each sample, normalized gene and transcript expression profiles were computed. The gene-level differential expression in different conditions were estimated using the log 2 transformed FPKM. While identifying differentially expressed genes DEGs , the uncorrected p -value of the test statistic and the FDR-adjusted p -value of the test statistic q -value were also calculated.
After Benjamini-Hochberg correction for multiple testing, any gene with a p -value higher than the FDR was considered significantly differentially expressed. The original contributions presented in the study are publicly available. SB has conceptualized, wrote the paper and supervised the study. JK performed the experiments, analyzed the data and edited the manuscript. YR performed the experiments and analyzed the data.
DR helped in analysis of flow cytometry data. NK gave critical inputs in editing of the paper. All authors contributed to the article and approved the submitted version. JK was supported by fellowship from the University Grants Commission.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The Translational Health Science and Technology Institute is acknowledged for providing all support for equipment and other infrastructure. Supplementary Figure 1. Subsequently, RNA from supernatant was extracted and purified. The normalized value of untreated mock was taken arbitrarily as 1 and those at day 3 and 6 expressed as fold-change with respect to that.
Supplementary Figure 2. The cells were permeabilized and immune-stained for intracellular DENV antigen and the fluorescence quantified by flow cytometry.
Supplementary Figure 3. A,B Normalized level of gene transcripts between uninfected and infected K, both of which were differentiated with 50 nM PMA for 6 days, were compared and the genes that were differentially regulated between these sets were tabulated. The top 10 terms identified in either group are represented as pie charts.
Supplementary Figure 4. Prediction of Transcription Factors regulating the expression of deregulated genes. Supplementary Figure 5. Supplementary Figure 6. Supplementary Figure 7. Supplementary Figure 8. Supplementary Table 1. List of genes that were differentially regulated by PMA-treatment for 6 days in uninfected K cells.
Supplementary Table 2. List of genes that were differentially regulated between uninfected and DENV-infected cells both of which have been differentiated by PMA-treatment for 6 days. Supplementary Table 3. Cecchetti, L. Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events. Blood , — Chaman, N. ERK2-pyruvate kinase axis permits phorbol myristate acetate-induced megakaryocyte differentiation in K cells.
Chen, S. ROS-mediated platelet generation: a microenvironment-dependent manner for megakaryocyte proliferation, differentiation, and maturation. Chen, Z. Expression analysis of primary mouse megakaryocyte differentiation and its application in identifying stage-specific molecular markers and a novel transcriptional target of NF-E2.
Choi, K. Caspase-dependent generation of reactive oxygen species in human astrocytoma cells contributes to resistance to TRAIL-mediated apoptosis. Death Differ. Clark, K. Colosetti, P. Autophagy is an important event for megakaryocytic differentiation of the chronic myelogenous leukemia K cell line. Autophagy 5, — Cullinan, S. PERK-dependent activation of Nrf2 contributes to redox homeostasis and cell survival following endoplasmic reticulum stress.
Daffis, S. Nature , —6. Thrombocytopenia in dengue: interrelationship between virus and the imbalance between coagulation and fibrinolysis and inflammatory mediators. Deutsch, V.
Megakaryocyte development and platelet production. Eliades, A. New roles for cyclin E in megakaryocytic polyploidization. Elshuber, S. Cleavage of protein prM is necessary for infection of BHK cells by tick-borne encephalitis virus. Fang, S. Recent advances in DENV receptors. World J. Gasiorek, J. Regulation and function of the NFE2 transcription factor in hematopoietic and non-hematopoietic cells. Life Sci. Ghosh, A. Effects of oxidative stress on protein translation: implications for cardiovascular diseases.
Golding, H. The phorbol ester phorbol myristate acetate inhibits human immunodeficiency virus type 1 envelope-mediated fusion by modulating an accessory component s in CD4-expressing cells. Hayes, J. The Nrf2 regulatory network provides an interface between redox and intermediary metabolism.
Trends Biochem. Henke, J. The induction of membrane structures may serve as a scaffold for anchoring the viral RC. The C-terminal regions of C, prM, and E contain hydrophobic amino acids that serve as signal sequences for insertion of the remaining protein into the ER membrane The NS3 protein acts, together with its cofactor NS2B, as the viral serine protease needed for polyprotein-processing through its N-terminal end 42 , However, a host cell signal peptidase mediates post-translational modifications on the NS4A-4B proteins The NS1 is a glycoprotein with two glycosylation sites that are conserved among flaviviruses.
It is synthesized in the ER as a hydrophilic monomer but exists as a more hydrophobic homodimer. The NS1 dimer is transported to the Golgi apparatus where it undergoes carbohydrate trimming The role of NS1 in virus replication is unknown but is believed to facilitate viral infection and DENV pathogenesis NS1 is in addition secreted from infected cells sNS1 and has been shown to be immunologically important 47 , Antibodies raised against sNS1 proteins have been proposed to cause endothelial dysfunction due to cross-reactivity to host proteins and endothelial cells Data indicate that sNS1 could be an important modulator of the complement pathway and is proposed to protect DENV from complement-dependent neutralization in solution Recent findings propose an inhibitory role in interferon IFN -mediated signal transduction.
Their hydrophobic nature potentially implicates them in proper localization of viral proteins and vRNA during replication and virion assembly. The cap structure is recognized by the host cell translational machinery. The NLS residues — interacts with the NS3 viral helicase and is recognized by cellular factors, allowing protein transport to the nucleus. Prior to secretion of new viral particles, the third structural protein pr M is processed into the mature M protein in the TGN by furin host protease It is believed that prM protects the E proteins from pH-induced reorganization and premature fusion during secretion; hence, the maturation event is necessary for infectivity 51 — Since laboratory-based dengue diagnosis is often unavailable at the time of care, the preliminary diagnosis relies on a combination of travel history and clinical symptoms.
Travel history provides key information that can rule out other potentially life-threatening diseases since the incubation period of DENV is less than 2 weeks A confirmed diagnosis for a DENV infection is established by culture of the virus, polymerase-chain reaction PCR , or serologic assays. There are, however, limitations with each test, and detection is based on different virological markers, namely infectious virus, vRNA, and DENV-specific antibodies, respectively.
Culturing the virus requires an acute patient serum with sufficient levels of virus, and the period when DENV can be successfully isolated in patient serum is short. Viremia peaks before the onset of symptoms, hence virus levels might drop significantly once the patient seeks medical care. Furthermore, rising levels of antibody interfere with virus culture already within a day or two after the subsidence of fever.
Apart from sample collection limitations, practical considerations limit the use of this method. Culture of the virus is both time- and labor intensive; infectious patient material must be kept cold, and a bio-safety level 3 laboratory is required, necessitating professional training of the personnel. These requirements limit the use of this diagnostic tool, especially in rural areas 4.
Specimens that may be suitable for virus isolation include acute phase serum; plasma or washed buffy coat from the patient; autopsy tissues from fatal cases, especially liver, spleen, lymph nodes, and thymus; and mosquitoes collected in nature Detection of vRNA from serum, plasma, or cells with PCR is based on DENV-specific oligonucleotide primers, and is fast and robust, although sensitive only in the very early stages of disease PCR is particularly useful in situations when virus culture has not been successful but nevertheless depends on sample collection during the symptomatic phase.
The third laboratory diagnostic option is not based on direct detection but on the presence of anti-DENV antibodies. Thus, it is not hindered by the limitations of virus culture and PCR, and the timing of sample collection can be more flexible. The immunoglobulins Ig are not easily inactivated and do not have the same strict requirements for low temperature as infectious virus specimen.
The assay techniques are relatively simple and there are commercial diagnostic kits available, whereof the assays based on IgM detection are the most commonly used in routine diagnostics 4. The major drawback with serological tests is the considerable risk for false-positive results due to potential cross-reactivity with other flaviviruses, for example, vaccination against Yellow fever virus YFV Due to the drawbacks of serological methods to reliably diagnose acute infections, alternative methods based on the detection of the viral NS1 protein have been developed.
NS1 can be found both membrane-associated inside the host cell and in a soluble, secreted form. The amount of secreted NS1 in patient serum correlates with viremia and DENV pathogenesis 46 , 57 — 60 , and the NS1 protein is detectable in serum by enzyme-linked immunosorbent assay ELISA from the first day of fever up to 9 days post-infection 46 , 61 — Several commercial NS1 antigen kits are available and are widely used in endemic as well as non-endemic countries. Vaccination must protect against all four serotypes without predisposing for antibody-dependent enhancement ADE and has proven difficult to design.
Nearly 80 years of vaccine-related research and development have passed, and over 25 unique DENV vaccine candidates have been tested in clinical trials during the past decade.
To be safe, a dengue vaccine must be functionally tetravalent, eliciting simultaneous protection against all four DENV serotypes. Hence, vaccination cannot proceed in an analogous sequential manner, and herein lies the greatest obstacle 67 — Live attenuated vaccines can induce durable humoral and cellular immune responses that mimic natural infection However, the viral replication must be discrete to preclude the development of significant illness.
It is expected that a live attenuated vaccine would be successful and require only a single dose since the vaccine against YFV is based on a live attenuated virus. However, it is more likely that booster immunizations will be required based on results from clinical trials using tetravalent formulations of live vaccine candidates aimed at eliciting neutralizing antibodies 72 — 75 The obvious challenge is when and how to boost; infectivity and immunogenicity in NHP models have not always clearly predicted the outcome of human trials 76 , Vaccination compliance may also be lower with a multi-dose vaccination strategy, especially in regions where resources are scarce, and at the same time where the need for a vaccine often is the most acute.
Currently, there are several dengue vaccine candidates at different stages of preclinical or clinical development. These multi-center studies in a variety of epidemiological settings will be important to obtain data regarding efficacy and safety, and will shed further light on the relationship between vaccine-induced immune responses and protection against clinical dengue disease There are in addition other live-attenuated, subunit, and DNA vaccine candidates at earlier stages of clinical development.
Other technological approaches include viral-vectored and virus-like particle vaccines, which currently are being tested in preclinical studies. It is hoped that clinical trials evaluating novel recombinant subunit proteins, DNA, and vectored vaccines would be initiated in the coming years. These approaches could be part of a prime-boost strategy, or stand-alone The use of different types of vaccines depends on the purpose of vaccination and target group reflecting the disease setting.
In endemic areas, there is an urgent need for routine immunization against dengue for infants and young children aged 1—3 years. A dengue vaccine would be coordinated with current childhood immunization schedules.
Due to the socioeconomic status of many endemic countries, this type of vaccine ought to be inexpensive. In contrast, a protective vaccine for international travel, seasonal work personnel, and military staff that visit or work in DENV endemic areas are more tolerant to increased cost. Vaccination in this case will need to be rapid.
In addition, antiviral drugs would be more potent in an outbreak situation than a vaccine when there is no time to complete a multi-dose immunization schedule spanning 6 months or more. Currently, vector control, regarded as both expensive and ineffective, is the only method for disease prevention 79 , In the absence of available vaccines and antiviral drugs against DENV infection, specific treatment for dengue patients consist primarily of supportive care including bed rest, antipyretics, and analgesics.
Urgent resuscitation with intravenous fluids to replace lost intravascular volume in DSS patients is a prerequisite; Ringer's lactate has been shown to be effective in moderately severe dengue, and starch or dextran have been suggested for more severe cases Aspirin and other salicylates should be avoided due to plasma leakage 6. The design of novel therapeutic approaches for dengue disease has focused on the various stages of the viral replication cycle.
The conformational changes of the E protein and its interaction with prM or M have been a major interest. These transition states present opportunities for antiviral targeting of the entry, assembly, or maturation steps of the virus life cycle. Targeting of mature virus entry into host cells is an extremely promising candidate since delivery of target compounds into the host cell during stages of fusion and maturation is significantly more challenging. Another approach to inhibit the structural changes of the E-prM protein interactions has been to synthesize peptides mimicking the pr peptide of the M protein, thereby preventing membrane fusion and release of newly synthesized virions.
The viral protease is another interesting target for antiviral discovery, since proteases are common to most viruses and generally important for efficient replication. Nucleoside analogues are usually prodrugs that need to be converted to their antiviral nucleotide metabolite forms.
Ribavirin depletes the nucleotide pool and thereby indirectly affects capping and polymerase activities of both cellular and viral proteins. In addition, ribavirin causes a more error-prone replication of several viral genomes. Despite successful in vivo results with several RNA viruses, ribavirin has a cytostatic effect in DENV-infected cells and has not been effective in animal models.
Nucleic acid—based therapies offer various alternatives. It has been used in therapeutic approaches for several infectious diseases, tumors, and metabolic disorders. These compounds act by forming a stable, sequence-specific duplex with RNA, thereby blocking access to target RNA by biomolecules required for replication. These compounds meet most of the requirements for an anti-DENV therapeutic; non-toxic, cheap, easy to administer, stable for months at variable temperatures, but remain to be tested in animal models.
Sulfated polysaccharides have been investigated for anti-DENV activity, although inconsistency in the activity results indicates that they need to be further tested both in vitro and in vivo The processing of N -linked oligosaccharides in the ER is important for viral glycoprotein maturation, and inhibition of glucosidase-mediated trimming affects the replication cycle of several enveloped viruses.
Nitric oxide NO is generated by macrophages, monocytes, dendritic cells DCs , and neutrophils; the same cells that are supposed to be the main sites of replication for DENV. Hence, there are multiple options for designing novel therapeutics for dengue disease. However, the main concern with most therapeutic approaches is that they are not validated for inhibitory effects on all four DENV serotypes. In addition, several studies have not been examined in an animal model, and several reported antivirals have been tested at only one time point, pre- or post-infection in tissue culture systems, and therefore need to be subjected to more diverse regimes, and different cell types.
DENV pathogenesis remains a challenging jigsaw puzzle with many pieces missing to understand the complex interplay of viral and host factors. Despite intensive research, it is not well understood. The severity of DENV infection is modulated by multiple risk factors such as age 83 , 84 , the genetic background of the host 85 , 86 , viral serotype 83 , 87 and genotype 88 , 89 , and secondary DENV infection by a heterologous serotype 85 , 90 — Finally, the virus serotype and genotype also influence the symptomatic picture of disease and outcome Fig.
These observations were initially based on epidemiological findings, but accumulating laboratory and experimental data have contributed to the recognition of DENV virulence as an important risk factor.
The complex interplay of risk factors for severe dengue disease can be illustrated as a triangular interplay dominated by the three main risk factors: host factors, preexisting DENV-specific antibodies mediating antibody-dependent enhancement ADE , and intrinsic virus features influencing strain virulence.
The exact contribution of each risk factor may vary from case to case. Apart from the influence of viral genetic determinants, the host's genetic background with varying polymorphisms might have important consequences for disease susceptibility Improvements in high-throughput genotyping of genetic polymorphisms have permitted a genome-wide approach to the investigation of host genetic susceptibility.
However, most studies have not attempted functional trials to try to link genetic association with any process in disease pathogenesis. Several serological studies of HLA class I alleles have been performed in ethnically and geographically distinct populations, and positive correlations of various HLA class I alleles with susceptibility to DHF have been found.
Since the Mexican Mestizo population and the Cuban population share the same Amerindian genetic background, it is possible that the identification of the same HLA class II allotype could explain the association to dengue disease protection.
The number of studies on polymorphisms within genes other than the HLA loci remains low. An increased association between severe dengue and bronchial asthma, diabetes mellitus, peptic ulcers, and sickle cell anaemia has been observed 94 , However, the impact of dengue on chronic diseases and other pathogens needs to be further investigated. Primary infections are supposed to cause mild disease in children, compared to secondary infections that tend to lead to severe dengue.
The greater relative prevalence of DSS in children relative to adults is believed to be due to the intrinsically more permeable vascular endothelium in children There is no clear consensus; studies conducted in South American countries have reported similar 84 as well as contradictory results indicating that adults are the most affected Antibodies against the viral surface E protein cross-react with plasminogen and have been associated with bleeding in acute DENV infection, and anti-DENV NS1 antibodies cross-react with host proteins and endothelial cells 49 , In addition, immune activation markers e.
The explanation lies within the cross-reactive antibodies raised after a primary DENV infection 90 , 92 , Serotype-specific antibodies confer life-long immunity to the homologous serotype, whereas cross-protection against heterologous serotypes last for 3—4 months.
This phenomenon is known as ADE The limited cross-protection between the four DENV serotypes has allowed them to coexist in the same or overlapping geographical areas. Thus, their antigenic uniqueness has implied an evolutionary advantage The net result will be a larger number of infected cells compared to the primary infection when there were no cross-reactive antibodies present, or compared to earlier after the primary infection when antibody levels are high enough to achieve neutralization of the heterologous virus.
In vitro studies indicate that non-neutralizing antibodies against the viral prM protein can potentially mediate ADE. These anti-prM antibodies are in addition non-neutralizing even at high concentrations 97 , The proposed hypothesis for prM-mediated ADE is based on the fact that the viral prM protein needs to be cleaved to render the virus infectious.
Hence, immature virus particles that would otherwise be non- or less-infectious are rendered infectious in combination with anti-prM antibodies that mediate ADE to infect new host cells The differences in DENV genotype could influence the pathogenic consequences, but a contributing risk factor is the progressive loss of heterotypic neutralizing antibodies However, primary infections in infants aged between 4 and 12 months of age run a higher risk of developing severe dengue due to maternally derived non-neutralizing antibodies.
The risk of severe dengue decreases after the age of 1 year as the concentration of cross-reactive antibodies declines 94 , , A higher viral burden elicits a greater host inflammatory response and increased plasma levels of proinflammatory cytokines. Thus, an increased infected cell mass would stimulate T-cell and cytokine responses that are proportional to the antigenic stimulus. Severe dengue with plasma leakage can occur in primary infection without ADE. In addition, by the time plasma leakage occurs, viral titers are several logs below peak levels, and there are patients with high viral titers that do not develop plasma leakage 59 , , Thus, increased viremia alone is not the direct cause of plasma leakage and other mechanisms are involved in the cytokine storm.
Furthermore, ADE is not a useful correlate of disease risk , ADE has dominated as the explanatory model for severe dengue disease in secondary infections.
Many parts of the world have become hyperendemic, implying that all four serotypes of DENV co-circulate in the same country, with the consequence that secondary infections are common scenarios. Studies from Thailand report that 0. In Cuba, The hypothesis that some DENV genotypes have greater virulence and epidemic potential than others was introduced during the s around the same time that the ADE phenomenon was coined — However, in contrast to the ADE hypothesis, experimental evidence for increased virulence was for long absent and, therefore, primarily based on epidemiological observations.
Recent work has shed light on this question and confirmed what Rosen, et al. It was also seen that the South-East Asian genotype infects and disseminates to the head tissue of Ae. Both traits likely enhanced the capacity to spread and displace endemic strains.
Based on the examples given, one hypothetical mechanism for increased virulence suggests that highly pathogenic DENV strains have been selected for enhanced ability to replicate in key human targets, such as macrophages and DCs 94 , Thus, virulent DENVs would produce more viruses per cell, resulting in higher viremia and inflammatory response, than with a low pathogenic strain 94 , Enhancement of virus replication following heterologous infection may favor coexistence of multiple serotypes.
If such enhancement also results in increased transmission, DENVs from different serotypes would benefit from prior and concurrent circulation of several serotypes in the same location It is still not known if the tendency of certain genotypes to cause severe disease results from greater intrinsic virulence, or if greater virulence is a result of enhanced infectivity in the presence of heterologous antibodies, or a combination of the two.
Determining whether DENVs differ in virulence, as well as identifying the genetic basis of such differences, is of fundamental importance. DENV infection is a systemic and dynamic disease with a wide clinical spectrum. Gross pathological findings in cases of DHF or DSS include hemorrhages in the skin, subcutaneous tissues, gastrointestinal tract, and heart Hemorrhage, dilatation and congestion of vessels, and edema of arterial walls are commonly found, and hemorrhagic manifestations in other organs combined with fluid accumulations in body cavities may be substantial , However, the underlying mechanisms of vascular leakage and hemorrhage are not well characterized.
Available data propose that the outcome of a DENV infection depends on a balance between favorable and unfavorable immune responses; the former providing control of viral replication, whereas the latter enhancing inflammatory and vascular permeability. The lack of reliable immunological markers for either protective or pathological immune responses to DENV and the lack of a suitable animal model for dengue disease hamper the understanding of dengue pathogenesis.
Insights into the immune response against DENV infection rely primarily on clinical and epidemiological studies. Identification of the primary target cells of DENV replication has proven to be extremely difficult.
Existing data are based on virus detection by immunohistochemical IHC analysis with antibodies against viral structural proteins, or by in situ hybridization to the positive-strand vRNA. However, it is difficult to prove direct infection of specific target cells by these methods as a positive signal could be due to virus endocytosed or phagocytosed by uninfected cells.
After inoculation by an infected mosquito, the initial round of viral replication is believed to occur in the subdermal Langerhans DCs — These infected cells become activated and migrate to draining lymph nodes Viral replication continues in still undefined cells in the lymph node. There is a general consensus that candidate cell types belong to the macrophage-monocyte lineage. Autopsies and human biopsies confirm that cells from the mononuclear phagocyte linage probably are the primary targets of DENV infection following initial dissemination from the local skin site.
Infiltrating mononuclear cells in affected tissues have been shown to contain DENV antigen , , and DENVs can occasionally be isolated from peripheral blood leukocyte fractions Similar observations have been made in rhesus macaques where DENV was recovered from leukocyte-rich tissues such as regional lymph nodes, systemic lymphatic tissues, and disseminated skin sites. Infection is amplified within the lymph nodes and viremia can be detected when the infectious virus enters the circulation via the efferent lymphatic system and thoracic duct.
Circulating monocytes in the blood are believed to be infected due to the viremia facilitating spread to secondary visceral organs where macrophages within the spleen, liver, and bone marrow are infected — There has been limited and inconsistent dissemination to solid organs ; DENV antigen has been detected in lymphocytes , , hepatocytes — , endothelium , , , , and cerebral neurons and astrocytes , There are in addition other studies with contradicting results where the same tissues have been examined without any detected DENV antigen , , A further controversy surrounds the role of endothelial cells as the target for DENV infection.
Severe dengue disease is characterized by systemic endothelial dysfunction accompanied by vascular leakage, even though destructive vascular lesions are generally absent in fatal cases Primary human endothelial cells and human endothelial cell lines are permissive for DENV infection 47 , , but endothelial infection, however, does not seem to be required for severe pathologic changes in individual tissues Their contribution in vivo remains to be established.
The presence of DENV antigens in various organs and cell types suggest that the host receptor s is broadly distributed.
Following DENV infection natural antibodies IgM , complement, and possibly NK cells control the initial levels of viremia and to certain extent tissue dissemination. Upon recognition by cytotoxic T lymphocytes, infected cells are targeted by the cellular immune system discussed below. The humoral immune response is hypothesized to be vital for controlling DENV infection and dissemination, and infection with one serotype provides long-lasting protection to that specific serotype homotypic immunity.
During virus replication, RNA synthesis is mediated by a dynamic and membrane-bound multi-protein assembly, named the replication complex RC.
The RC is composed of both viral and host-cell proteins that assemble within vesicles composed of the endoplasmic reticulum membrane, near the nucleus.
At the heart of the flavivirus RC lies NS4B, a viral integral membrane protein that plays a role in virulence and in down-regulating the innate immune response.
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