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1.
Nutrients ; 13(12)2021 Dec 16.
Article in English | MEDLINE | ID: mdl-34960061

ABSTRACT

Dengue fever (DF) is a mosquito-borne disease still with no effective treatment or vaccine available. A randomized, placebo-controlled, double-blinded, parallel-group trial was undertaken to evaluate the efficacy of oral intake of Lactococcus lactis strain plasma (LC-Plasma) on the presentation and severity of DF-like symptoms among healthy volunteers. Study participants (320) were assigned into two groups, and consumed either placebo or LC-Plasma tablets (approximately 100 billion cells/day) for 8 weeks. The clinical symptoms of DF were self-recorded through questionnaires, and exposure to DENV was determined by serum antibody and/or DENV antigen tests. No significant differences between groups were observed for exposure to DENV, or the symptomatic ratio. Results obtained showed that participants from the LC-Plasma group reported a significant reduction in the cumulative incidence days of DF-like symptoms, which include fever (p < 0.001), muscle pain (p < 0.005), joint pain (p < 0.001), and pain behind the eyes (p < 0.001), compared to that of the placebo group. Subgroup analysis revealed a significantly (p < 0.05) reduced severity score in the LC-Plasma group when study sites were separately analyzed. Overall, our findings suggest that LC-Plasma supplementation reduces the cumulative days with DF-like symptoms, and the severity of the symptoms. Daily oral intake of LC-Plasma, hence, is shown to mitigate the DF-like symptoms.


Subject(s)
Dengue Virus/immunology , Dengue/prevention & control , Lactococcus lactis/immunology , Probiotics/administration & dosage , Administration, Oral , Adult , Dengue/epidemiology , Dengue/virology , Double-Blind Method , Female , Humans , Malaysia/epidemiology , Male , Treatment Outcome
2.
Emerg Microbes Infect ; 11(1): 227-239, 2022 Dec.
Article in English | MEDLINE | ID: mdl-34931940

ABSTRACT

Dengue is caused by four genetically distinct viral serotypes, dengue virus (DENV) 1-4. Following transmission by Aedes mosquitoes, DENV can cause a broad spectrum of clinically apparent disease ranging from febrile illness to dengue hemorrhagic fever and dengue shock syndrome. Progress in the understanding of different dengue serotypes and their impacts on specific host-virus interactions has been hampered by the scarcity of tools that adequately reflect their antigenic and genetic diversity. To bridge this gap, we created and characterized infectious clones of DENV1-4 originating from South America, Africa, and Southeast Asia. Analysis of whole viral genome sequences of five DENV isolates from each of the four serotypes confirmed their broad genetic and antigenic diversity. Using a modified circular polymerase extension reaction (CPER), we generated de novo viruses from these isolates. The resultant clones replicated robustly in human and insect cells at levels similar to those of the parental strains. To investigate in vivo properties of these genetically diverse isolates, representative viruses from each DENV serotype were administered to NOD Rag1-/-, IL2rgnull Flk2-/- (NRGF) mice, engrafted with components of a human immune system. All DENV strains tested resulted in viremia in humanized mice and induced cellular and IgM immune responses. Collectively, we describe here a workflow for rapidly generating de novo infectious clones of DENV - and conceivably other RNA viruses. The infectious clones described here are a valuable resource for reverse genetic studies and for characterizing host responses to DENV in vitro and in vivo.


Subject(s)
Dengue Virus/genetics , Dengue Virus/immunology , Dengue/virology , Aedes/physiology , Aedes/virology , Animals , Antibodies, Viral/immunology , Antigenic Variation , Dengue/genetics , Dengue/immunology , Dengue/transmission , Dengue Virus/classification , Dengue Virus/isolation & purification , Genetic Variation , Humans , Immunoglobulin M/immunology , Mice , Mice, Knockout , Reverse Genetics , Serogroup
3.
PLoS One ; 16(12): e0261412, 2021.
Article in English | MEDLINE | ID: mdl-34929011

ABSTRACT

BACKGROUND: Dengue is the most rapidly spreading mosquito-borne viral disease of humans worldwide, including southeast Asia region. This review provides a comprehensive overview of questionnaire-related dengue studies conducted in the Philippines and evaluates their reliability and validity in these surveys. METHODS: A review protocol constructed by a panel of experienced academic reviewers was used to formulate the methodology, research design, search strategy and selection criteria. An extensive literature search was conducted between March-June 2020 in various major electronic biomedical databases including PubMed, EMBASE, MEDLINE and ScienceDirect. A systematic review and meta-analysis (PRISMA) were selected as the preferred item reporting method. RESULTS: Out of a total of 34 peer-reviewed dengue-related KAP studies that were identified, 15 published from 2000 to April 2020 met the inclusion criteria. Based on the meta-analysis, a poor mean score was obtained for each of knowledge (68.89), attitude (49.86) and preventive practice (64.69). Most respondents were equipped with a good knowledge of the major clinical signs of dengue. Worryingly, 95% of respondents showed several negative attitudes towards dengue prevention, claiming that this was not possible and that enacting preventive practices was not their responsibility. Interestingly, television or radio was claimed as the main source of gaining dengue information (range 50-95%). Lastly, only five articles (33.3%) piloted or pretested their questionnaire before surveying, of which three reported Cronbach's alpha coefficient (range 0.70 to 0.90). CONCLUSION: This review indicates that to combat the growing public health threat of dengue to the Philippines, we need the active participation of resident communities, full engagement of healthcare personnel, promotion of awareness campaigns, and access to safe complementary and alternative medicines. Importantly, the psychometric properties of each questionnaire should be assessed rigorously.


Subject(s)
Dengue Virus/isolation & purification , Dengue/prevention & control , Health Knowledge, Attitudes, Practice , Health Personnel/psychology , Health Services Research/statistics & numerical data , Surveys and Questionnaires/statistics & numerical data , Dengue/etiology , Dengue/psychology , Dengue/virology , Humans , Philippines/epidemiology , Reproducibility of Results
4.
J Gen Virol ; 102(10)2021 10.
Article in English | MEDLINE | ID: mdl-34665110

ABSTRACT

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip-/-) showed enhanced DENV infection, accompanied by increased IFN-ß and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip-/- mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip-/- compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-ß, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip-/- mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip-/- DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-ß, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip-/- mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.


Subject(s)
Antiviral Agents , Dengue Virus/immunology , Dengue Virus/physiology , Dengue/immunology , Dengue/virology , Immunity, Innate , Proteins/physiology , Animals , Brain/immunology , Brain/virology , Cells, Cultured , Inflammation , Interferon Regulatory Factor-7/genetics , Interferon-beta/biosynthesis , Interferon-beta/genetics , Macrophages/immunology , Macrophages/metabolism , Mice , Proteins/genetics , Virus Replication
5.
Sci Rep ; 11(1): 19713, 2021 10 05.
Article in English | MEDLINE | ID: mdl-34611200

ABSTRACT

The novel coronavirus disease 2019 (COVID-19) presents with non-specific clinical features. This may result in misdiagnosis or delayed diagnosis, and lead to further transmission in the community. We aimed to derive early predictors to differentiate COVID-19 from influenza and dengue. The study comprised 126 patients with COVID-19, 171 with influenza and 180 with dengue, who presented within 5 days of symptom onset. All cases were confirmed by reverse transcriptase polymerase chain reaction tests. We used logistic regression models to identify demographics, clinical characteristics and laboratory markers in classifying COVID-19 versus influenza, and COVID-19 versus dengue. The performance of each model was evaluated using receiver operating characteristic (ROC) curves. Shortness of breath was the strongest predictor in the models for differentiating between COVID-19 and influenza, followed by diarrhoea. Higher lymphocyte count was predictive of COVID-19 versus influenza and versus dengue. In the model for differentiating between COVID-19 and dengue, patients with cough and higher platelet count were at increased odds of COVID-19, while headache, joint pain, skin rash and vomiting/nausea were indicative of dengue. The cross-validated area under the ROC curve for all four models was above 0.85. Clinical features and simple laboratory markers for differentiating COVID-19 from influenza and dengue are identified in this study which can be used by primary care physicians in resource limited settings to determine if further investigations or referrals would be required.


Subject(s)
COVID-19/pathology , Dengue/pathology , Influenza, Human/pathology , Adult , Area Under Curve , COVID-19/complications , COVID-19/virology , Cohort Studies , Dengue/complications , Dengue/virology , Diagnosis, Differential , Diarrhea/etiology , Female , Fever/etiology , Humans , Influenza, Human/complications , Influenza, Human/virology , Lymphocyte Count , Male , Middle Aged , Platelet Count , RNA, Viral/analysis , RNA, Viral/metabolism , ROC Curve , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Vomiting/etiology , Young Adult
6.
Nat Commun ; 12(1): 5374, 2021 09 10.
Article in English | MEDLINE | ID: mdl-34508072

ABSTRACT

The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and Zika virus, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision-guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate flightless females and sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and even eliminate mosquito populations. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.


Subject(s)
Aedes/virology , Infertility, Male/veterinary , Mosquito Control/methods , Mosquito Vectors/virology , Aedes/genetics , Animals , Animals, Genetically Modified , Arboviruses , Chikungunya Fever/prevention & control , Chikungunya Fever/transmission , Chikungunya Fever/virology , Dengue/prevention & control , Dengue/transmission , Dengue/virology , Female , Humans , Infertility, Male/genetics , Male , Models, Biological , Mosquito Vectors/genetics , Yellow Fever/prevention & control , Yellow Fever/transmission , Yellow Fever/virology , Zika Virus , Zika Virus Infection/prevention & control , Zika Virus Infection/transmission , Zika Virus Infection/virology
7.
Sci Rep ; 11(1): 18000, 2021 09 09.
Article in English | MEDLINE | ID: mdl-34504185

ABSTRACT

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer-antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.


Subject(s)
Antibodies, Viral/immunology , Antibody Affinity , Aptamers, Nucleotide/immunology , Dengue Virus/immunology , Dengue/immunology , Immunoglobulin G/immunology , Serogroup , Serologic Tests/methods , Viral Nonstructural Proteins/immunology , Antibodies, Viral/blood , Antigens, Viral/immunology , Cross Reactions , Dengue/blood , Dengue/diagnosis , Dengue/virology , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoglobulin G/blood , Sensitivity and Specificity , Zika Virus/immunology , Zika Virus Infection/blood , Zika Virus Infection/diagnosis , Zika Virus Infection/immunology , Zika Virus Infection/virology
8.
J Adv Res ; 32: 37-44, 2021 09.
Article in English | MEDLINE | ID: mdl-34484824

ABSTRACT

Introduction: Dengue and Malaria are the most important mosquito-borne viral diseases affecting humans. Fever is transmitted between human hosts by infected female aedes mosquitoes. The modeling study of viral infections is very useful to show how the virus replicates in an infected individual and how the human antibody response acts to control that replication, which antibody playing a key role in controlling infection. Objectives: Optimal control of a novel variable-order nonlinear model of dengue virus is studied in the present work. Bang-bang control is suggested to minimize the viral infection as well as quick clearance of the virus from the host. Necessary conditions for the control problem are given. The variable-order derivatives are given in the sense of Caputo. Moreover, the parameters of the proposed model are dependent on the same variable-order fractional power. Two numerical schemes are constructed for solving the optimality systems. Comparative studies and numerical simulations are implemented. The variable-order fractional derivative can be describe the effects of long variable memory of time dependent systems than the integer order and fractional order derivatives. Methods: Both the nonstandard generalized fourth order Runge-Kutta and the nonstandard generalized Euler methods are presented. Results: We have successfully applied a kind of Pontryagin's maximum principle with bang-bang control and were able to reduce the viraemia level by adding the dose of DI particles. The nonstandard generalized fourth order Runge-Kutta method has the best results than nonstandard generalized Euler method. Conclusion: The combination of the variable-order fractional derivative and bang-bang control in the Dengue mathematical model improves the dynamics of the model. The nonstandard generalized Euler method and the nonstandard generalized fourth order Runge-Kutta method can be used to study the variable order fractional optimal control problem simply.


Subject(s)
Dengue Virus , Dengue/virology , Models, Theoretical , Aedes , Animals , Female , Humans , Viremia/virology , Virus Replication
9.
Viruses ; 13(8)2021 08 18.
Article in English | MEDLINE | ID: mdl-34452505

ABSTRACT

Viral infection is a global public health threat causing millions of deaths. A suitable small animal model is essential for viral pathogenesis and host response studies that could be used in antiviral and vaccine development. The tree shrew (Tupaia belangeri or Tupaia belangeri chinenesis), a squirrel-like non-primate small mammal in the Tupaiidae family, has been reported to be susceptible to important human viral pathogens, including hepatitis viruses (e.g., HBV, HCV), respiratory viruses (influenza viruses, SARS-CoV-2, human adenovirus B), arboviruses (Zika virus and dengue virus), and other viruses (e.g., herpes simplex virus, etc.). The pathogenesis of these viruses is not fully understood due to the lack of an economically feasible suitable small animal model mimicking natural infection of human diseases. The tree shrew model significantly contributes towards a better understanding of the infection and pathogenesis of these important human pathogens, highlighting its potential to be used as a viable viral infection model of human viruses. Therefore, in this review, we summarize updates regarding human viral infection in the tree shrew model, which highlights the potential of the tree shrew to be utilized for human viral infection and pathogenesis studies.


Subject(s)
Disease Models, Animal , Tupaia , Virus Diseases , Adenoviridae Infections/immunology , Adenoviridae Infections/virology , Animals , COVID-19/virology , Dengue/immunology , Dengue/pathology , Dengue/virology , HIV Infections/virology , Hepatitis B/immunology , Hepatitis B/virology , Hepatitis C/immunology , Hepatitis C/pathology , Hepatitis C/virology , Herpes Simplex/pathology , Herpes Simplex/virology , Humans , Influenza, Human/immunology , Influenza, Human/virology , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/virology , Zika Virus Infection/immunology , Zika Virus Infection/pathology , Zika Virus Infection/virology
10.
J Gen Virol ; 102(8)2021 08.
Article in English | MEDLINE | ID: mdl-34410905

ABSTRACT

The capsid protein (C) of dengue virus is required for viral infectivity as it packages viral RNA genome into infectious particles. C exists as a homodimer that forms via hydrophobic interactions between the α2 and α4 helices of monomers. To identify C region(s) important for virus particle production, a complementation system was employed in which single-round infectious particles are generated by trans-encapsidation of a viral C-deleted genome by recombinant C expressed in mosquito cells. Mutants harbouring a complete α3 deletion, or a dual Ile65-/Trp69-to-Ala substitution in the α3 helix, exhibited reduced production of infectious virus. Unexpectedly, higher proportions of oligomeric C were detected in cells expressing both mutated forms as compared with the wild-type counterpart, indicating that the α3 helix, through its internal hydrophobic residues, may down-modulate oligomerization of C during particle formation. Compared with wild-type C, the double Ile65-/Trp69 to Ala mutations appeared to hamper viral infectivity but not C and genomic RNA incorporation into the pseudo-infectious virus particles, suggesting that increased C oligomerization may impair DENV replication at the cell entry step.


Subject(s)
Capsid Proteins , Capsid/metabolism , Dengue Virus/metabolism , Dengue/virology , Aedes , Amino Acid Sequence , Animals , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Chlorocebus aethiops , Humans , Vero Cells , Virus Assembly , Virus Replication
11.
Viruses ; 13(7)2021 07 12.
Article in English | MEDLINE | ID: mdl-34372551

ABSTRACT

BACKGROUND: In 2015-2016, a large Zika virus (ZIKV) outbreak occurred in the Americas. Although the exact ZIKV antibody kinetics after infection are unknown, recent evidence indicates the rapid waning of ZIKV antibodies in humans. Therefore, we aimed to determine the levels of ZIKV antibodies more than three years after a ZIKV infection. METHODS: We performed ZIKV virus neutralization tests (VNT) and a commercial ZIKV non-structural protein 1 (NS1) IgG ELISA in a cohort of 49 participants from Suriname who had a polymerase-chain-reaction-confirmed ZIKV infection more than three years ago. Furthermore, we determined the presence of antibodies against multiple dengue virus (DENV) antigens. RESULTS: The ZIKV seroprevalence in this cohort, assessed with ZIKV VNT and ZIKV NS1 IgG ELISA, was 59.2% and 63.3%, respectively. There was, however, no correlation between these two tests. Furthermore, we did not find evidence of a potential negative influence of DENV immunity on ZIKV antibody titers. CONCLUSIONS: ZIKV seroprevalence, assessed with two commonly used serological tests, was lower than expected in this cohort of participants who had a confirmed previous ZIKV infection. This can have implications for future ZIKV seroprevalence studies and possibly for the duration of immunological protection after a ZIKV infection.


Subject(s)
Antibodies, Neutralizing/analysis , Zika Virus Infection/immunology , Zika Virus/immunology , Adult , Antibodies, Neutralizing/immunology , Antibodies, Viral/analysis , Antibodies, Viral/immunology , Cohort Studies , Cross Reactions/immunology , Dengue/virology , Dengue Virus/immunology , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Male , Middle Aged , Neutralization Tests/methods , Seroepidemiologic Studies , Serologic Tests/methods , Suriname , Zika Virus/pathogenicity , Zika Virus Infection/metabolism , Zika Virus Infection/virology
12.
Viruses ; 13(7)2021 07 17.
Article in English | MEDLINE | ID: mdl-34372598

ABSTRACT

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.


Subject(s)
Dengue Virus/chemistry , Dengue Virus/pathogenicity , Viral Nonstructural Proteins/metabolism , Animals , Cell Line , Chlorocebus aethiops , Chromatography, Liquid , Dengue/virology , Dengue Virus/genetics , Hep G2 Cells , Humans , Kinetics , Phosphorylation , Protein Binding , Sequence Analysis, Protein , Tandem Mass Spectrometry , Vero Cells , Viral Nonstructural Proteins/genetics , Virus Replication
13.
Viruses ; 13(7)2021 07 19.
Article in English | MEDLINE | ID: mdl-34372606

ABSTRACT

As demonstrated with the novel coronavirus pandemic, rapid and accurate diagnosis is key to determine the clinical characteristic of a disease and to improve vaccine development. Once the infected person is identified, hematological findings may be used to predict disease outcome and offer the correct treatment. Rapid and accurate diagnosis and clinical parameters are pivotal to track infections during clinical trials and set protection status. This is also applicable for re-emerging diseases like dengue fever, which causes outbreaks in Asia and Latin America every 4 to 5 years. Some areas in the US are also endemic for the transmission of dengue virus (DENV), the causal agent of dengue fever. However, significant number of DENV infections in rural areas are diagnosed solely by clinical and hematological findings because of the lack of availability of ELISA or PCR-based tests or the infrastructure to implement them in the near future. Rapid diagnostic tests (RDT) are a less sensitive, yet they represent a timely way of detecting DENV infections. The purpose of this study was to determine whether there is an association between hematological findings and the probability for an NS1-based DENV RDT to detect the DENV NS1 antigen. We also aimed to describe the hematological parameters that are associated with the diagnosis through each test.


Subject(s)
COVID-19/diagnosis , COVID-19/epidemiology , Dengue/diagnosis , Adolescent , Adult , Asia/epidemiology , Child , Child, Preschool , Colombia/epidemiology , Dengue/virology , Dengue Virus/isolation & purification , Diagnostic Tests, Routine/methods , Enzyme-Linked Immunosorbent Assay , Female , Humans , Infant , Male , Pandemics , Polymerase Chain Reaction , Reagent Kits, Diagnostic , SARS-CoV-2/isolation & purification , Young Adult
14.
J Gen Virol ; 102(8)2021 08.
Article in English | MEDLINE | ID: mdl-34342561

ABSTRACT

Dengue virus (DENV) is the most prevalent pathogen of the Flaviviridae family. Due to the considerable increase in DENV incidence and spread, symptoms such as CNS involvement have increased. Heparan sulphate (HS) was the first molecule identified as an adhesion factor for DENV in mammalian cells. Viral phenotypes with different HS interactions are associated with various clinical symptoms, including neurological alterations. Here, using in silico analyses, in vitro studies, and the in vivo mouse model, we characterized two natural circulating DENV3 genotype I (GI) lineage 1 (L1) in Brazil-DENV3 MG-20 (from Minas Gerais) and DENV3 PV_BR (from Rondônia) that present divergent neurovirulent profiles and sensitivity to sulphated molecules. We identified substitutions at the viral envelope (E) in positions 62 and 123 as likely responsible for the differences in neurovirulence. The E62K and E123Q substitutions in DENV3 MG-20 and DENV3 PV_BR, respectively, greatly influenced in silico electrostatic density and heparin docking results. In vivo, mice inoculated with DENV3 MG-20 died, but not those infected with DENV3 PV_BR. The clinical symptoms, such as paralysis of the lower limbs and meningoencephalitis, and histopathology, also differed between the inoculated groups. In vitro heparin and heparinases assays further demonstrated the biological impact of these substitutions. Other characteristics that have been previously associated with alterations in cell tropism and neurovirulence, such as changes in the size of lysis plaques and differences in cytopathic effects in glioblastoma cells, were also observed.


Subject(s)
Dengue Virus/classification , Dengue Virus/genetics , Dengue/virology , Genotype , Heparitin Sulfate/metabolism , Viral Envelope Proteins/chemistry , Animals , Binding Sites , Brain/pathology , Cell Communication , Cell Line , Dengue/pathology , Dengue Virus/physiology , Disease Models, Animal , Female , Heparin , Host-Pathogen Interactions/physiology , Humans , Mice , Mice, Inbred BALB C , Molecular Docking Simulation , Phenotype , Phylogeny , Protein Conformation , Viral Envelope Proteins/classification , Viral Envelope Proteins/genetics , Virulence , Virus Attachment
15.
Front Immunol ; 12: 677874, 2021.
Article in English | MEDLINE | ID: mdl-34335578

ABSTRACT

Background: Early biomarkers of progression to severe dengue are urgently required to enable effective patient management and control treatment costs. Innate immune cells, which comprise the earliest responders to infection and along with the cytokines and chemokines they secrete, play a vital role in orchestrating the subsequent adaptive immune response and have been implicated in the enhancement of infection and "cytokine storm" associated with dengue severity. We investigated the early innate immune cytokine profile of dengue patients during acute phase of disease in a prospective blinded study that included subjects with acute dengue and febrile controls from four major hospitals in Bengaluru, India along with healthy controls. We used intracellular cytokine staining and flow cytometry to identify innate immune biomarkers that can predict progression to severe dengue. Results: Dengue infection resulted in enhanced secretion of multiple cytokines by all queried innate immune cell subsets, dominated by TNF-α from CD56+CD3+ NKT cells, monocyte subsets, and granulocytes along with IFN-γ from CD56+CD3+ NKT cells. Of note, significantly higher proportions of TNF-α secreting granulocytes and monocyte subsets at admission were associated with mild dengue and minimal symptoms. Dengue NS1 antigenemia used as a surrogate of viral load directly correlated with proportion of cytokine-secreting innate immune cells and was significantly higher in those who went on to recover with minimal symptoms. In patients with secondary dengue or those with bleeding or elevated liver enzymes who revealed predisposition to severe outcomes, early activation as well as efficient downregulation of innate responses were compromised. Conclusion: Our findings suggested that faulty/delayed kinetics of innate immune activation and downregulation was a driver of disease severity. We identified IFN-γ +CD56+CD3+ NKT cells and IL-6+ granulocytes at admission as novel early biomarkers that can predict the risk of progression to severity (composite AUC = 0.85-0.9). Strong correlations among multiple cytokine-secreting innate cell subsets revealed that coordinated early activation of the entire innate immune system in response to dengue virus infection contributed to resolution of infection and speedy recovery.


Subject(s)
Cytokines/blood , Dengue Virus/genetics , Dengue Virus/immunology , Dengue/blood , Dengue/immunology , Granulocytes/immunology , Immunity, Innate , Natural Killer T-Cells/immunology , Severity of Illness Index , Adolescent , Adult , Aged , Biomarkers/blood , Cytokine Release Syndrome/immunology , Cytokines/biosynthesis , Cytokines/immunology , Dengue/epidemiology , Dengue/virology , Female , Humans , India/epidemiology , Male , Middle Aged , Prospective Studies , Young Adult
16.
PLoS Negl Trop Dis ; 15(7): e0009641, 2021 07.
Article in English | MEDLINE | ID: mdl-34329306

ABSTRACT

Concerns have arisen that pre-existing immunity to dengue virus (DENV) could enhance Zika virus (ZIKV) disease, due to the homology between ZIKV and DENV and the observation of antibody-dependent enhancement (ADE) among DENV serotypes. To date, no study has examined the impact of pre-existing DENV immunity on ZIKV pathogenesis during pregnancy in a translational non-human primate model. Here we show that macaques with a prior DENV-2 exposure had a higher burden of ZIKV vRNA in maternal-fetal interface tissues as compared to DENV-naive macaques. However, pre-existing DENV immunity had no detectable impact on ZIKV replication kinetics in maternal plasma, and all pregnancies progressed to term without adverse outcomes or gross fetal abnormalities detectable at delivery. Understanding the risks of ADE to pregnant women worldwide is critical as vaccines against DENV and ZIKV are developed and licensed and as DENV and ZIKV continue to circulate.


Subject(s)
Dengue Virus , Dengue/immunology , Maternal-Fetal Exchange , Zika Virus Infection/pathology , Zika Virus , Animals , Antibodies, Viral/blood , Antibodies, Viral/metabolism , Antigens, Viral , Dengue/virology , Female , Infectious Disease Transmission, Vertical , Placenta , Pregnancy , RNA, Viral , Virus Replication
17.
Parasit Vectors ; 14(1): 376, 2021 Jul 26.
Article in English | MEDLINE | ID: mdl-34311776

ABSTRACT

BACKGROUND: Dengue fever is the most rapidly spreading mosquito-borne viral disease globally. More than 2.5 billion people live in dengue-endemic areas. Previous studies suggested an interrelationship between diabetes mellitus (DM) and dengue hemorrhagic fever (DHF). Conversely, glycolysis is a critical metabolic pathway for optimal dengue virus (DENV) replication. However, little is known concerning the effect of glucose on DENV replication in mosquitoes. In this study, we investigated the impact of glucose on DENV replication in mosquitoes Aedes aegypti. METHODS: Mosquitoes (Ae. aegypti UGAL/Rockefeller strain) were orally infected with DENV (serotype 2, 16681 strain) through infectious blood feeding. The DENV infection and transmission rates were determined by examining mosquito bodies and saliva, respectively, for DENV positivity at different time points after infection. In addition, a reverse genetic approach was applied by introducing double-stranded RNA against genes of interest into the mosquitoes to inhibit gene expression. RESULTS: Our data revealed a significant increase of DENV genome levels in mosquitoes consuming an infectious blood meal supplemented with glucose, suggesting that blood glucose is an important factor for viral replication. Interestingly, a significant increase of DENV E protein levels was detected in the saliva 4 days faster in mosquitoes that consumed infectious blood meals supplemented with glucose than in those consuming infectious blood meals alone. Furthermore, we perform RNAi to silence AKT or TOR and investigate the molecular mechanism regulating the glucose-mediated enhancement of viral replication. Silencing of AKT or TOR significantly reduced DENV titers in mosquitoes. CONCLUSIONS: This study suggested that blood glucose is beneficial to DENV replication and that it facilitates virus transmission in mosquitoes via AKT and TOR signaling. Therefore, our results strengthen our understanding of dengue fever and DM co-morbidity and possibly reveal new targets for specific antiviral therapies.


Subject(s)
Aedes/virology , Blood Glucose , Dengue Virus/drug effects , Dengue/transmission , Mosquito Vectors/virology , Signal Transduction/drug effects , Animals , Dengue/virology , Dengue Virus/physiology , Female , Humans , RNA Interference , Serogroup , Virus Replication/drug effects
18.
PLoS Pathog ; 17(7): e1008603, 2021 07.
Article in English | MEDLINE | ID: mdl-34310658

ABSTRACT

Dengue virus (DENV) is a mosquito-borne pathogen that causes a spectrum of diseases including life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage is a common clinical crisis in DHF/DSS patients and highly associated with increased endothelial permeability. The presence of vascular leakage causes hypotension, circulatory failure, and disseminated intravascular coagulation as the disease progresses of DHF/DSS patients, which can lead to the death of patients. However, the mechanisms by which DENV infection caused the vascular leakage are not fully understood. This study reveals a distinct mechanism by which DENV induces endothelial permeability and vascular leakage in human endothelial cells and mice tissues. We initially show that DENV2 promotes the matrix metalloproteinase-9 (MMP-9) expression and secretion in DHF patients' sera, peripheral blood mononuclear cells (PBMCs), and macrophages. This study further reveals that DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 facilitates the MMP-9 enzymatic activity, which alters the adhesion and tight junction and vascular leakage in human endothelial cells and mouse tissues. Moreover, NS1 recruits MMP-9 to interact with ß-catenin and Zona occludens protein-1/2 (ZO-1 and ZO-2) and to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mouse tissues. Thus, we reveal that DENV NS1 and MMP-9 cooperatively induce vascular leakage by impairing endothelial cell adhesion and tight junction, and suggest that MMP-9 may serve as a potential target for the treatment of hypovolemia in DSS/DHF patients.


Subject(s)
Dengue/pathology , Endothelial Cells/metabolism , Matrix Metalloproteinase 9/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Capillary Permeability/physiology , Cell Adhesion/physiology , Dengue/metabolism , Dengue/virology , Dengue Virus/metabolism , Humans , Mice , Tight Junctions/metabolism
19.
J Vector Borne Dis ; 57(2): 153-160, 2020.
Article in English | MEDLINE | ID: mdl-34290160

ABSTRACT

BACKGROUND & OBJECTIVES: Aedes aegypti is the most prominent vector for dengue virus worldwide. Accurate identification of the species and understanding its colonization pattern are essential prerequisites in vector control. Thus, the present study was aimed to genetically characterize Ae. aegypti mosquitoes collected from different regions of Sri Lanka based on mitochondrial COI gene. METHODS: Thirty-three Ae. aegypti larval samples were collected from 19 districts. A 735bp region of the mitochondrial COI gene was amplified and analyzed for genetic diversity indices. Phylogenetic trees were constructed using Sri Lankan samples and also including mosquito samples reported from other parts of the world. RESULTS: High genetic diversity was observed within the samples analysed (gene diversity: 0.949; average number of nucleotide differences: 6.371). There were 20 haplotypes presented within the 19 localities investigated. The phylogenetic tree derived two main clades. However, no distinguishable clustering pattern was observed in the phylogenetic tree except for the districts in the northern corner indicating extensive admixing among different populations. When samples from other countries were included in the phylogenetic tree, Anuradhapura, and Mannar samples were clustered together with samples from India, Venezuela, USA, Portugal and Cambodia while Rathnapura was clustered with Bolivia and France. INTERPRETATION & CONCLUSION: Our results suggest that Sri Lanka has undergone multiple invasions of Ae. aegypti from various parts of the world over an extensive period. Further, the mosquito control campaigns had not caused a significant effect on the Ae. aegypti populations which is existing in mutation-drift equilibrium.


Subject(s)
Aedes/genetics , Dengue/transmission , Genetic Variation , Mosquito Vectors/genetics , Aedes/virology , Animals , Dengue/virology , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Genetics, Population , Haplotypes , Introduced Species , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Mosquito Control , Mosquito Vectors/virology , Phylogeny , Sri Lanka
20.
J Vector Borne Dis ; 57(2): 176-181, 2020.
Article in English | MEDLINE | ID: mdl-34290163

ABSTRACT

BACKGROUND & OBJECTIVES: The state of Punjab, India is highly endemic for dengue fever as high number of confirmed dengue cases have been reported since 2013. A better understanding of vectors distribution and their seasonal variation is necessary to control the disease. Therefore, the present study was conducted in both rural and urban areas of 11 out of 22 districts of Punjab to highlight seasonal prevalence of Aedes vector mosquitoes. METHODS: Entomological surveys were carried out in different seasons and all kinds of indoor and outdoor breeding habitats were examined and Aedes immatures were collected. The Stegomyia indices were calculated and compared from urban and rural areas in different seasons. RESULTS: Both vectors of dengue, i.e. Aedes aegypti and Ae. albopictus were recorded to be prevalent. Ae. aegypti mosquitoes were observed in all districts surveyed while Ae. albopictus were found only in seven districts of Punjab. The Stegomyia indices were significantly high during monsoon as compared to pre- and post- monsoon periods. Occurrence of dengue cases were found to be correlated with the Stegomyia indices. INTERPRETATION & CONCLUSION: This is the first detailed study of prevalence of dengue mosquito vectors in Punjab showing the presence of Ae. aegypti and Ae. albopictus in both urban and rural areas of the state, thereby demonstrating wide distribution of this vector. Different breeding habitats identified in the study should be subjected to targeted intervention such as source reduction in order to achieve effective control of dengue cases.


Subject(s)
Aedes/physiology , Dengue Virus/physiology , Dengue/epidemiology , Mosquito Vectors/physiology , Aedes/virology , Animals , Dengue/transmission , Dengue/virology , Ecosystem , Entomology , Female , Humans , India/epidemiology , Larva , Mosquito Vectors/virology , Prevalence , Seasons
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