Defective interfering viral particles in acute dengue infections

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.     

Plasma Levels of Inter-α Inhibitor Proteins in Children with Acute Dengue Virus Infection

Background

Inter-α inhibitor proteins (IaIp) belong to a family of protease inhibitors that are involved in the haemostatic and the vascular system. Dengue viruses (DENV) infections are characterized by coagulopathy and increased vascular permeability. In this study we measured the concentration of IaIp during DENV infections and evaluated its potential as a biomarker.

Methods and Findings

Concentrations of IaIp were measured in patients with acute DENV infections using a quantitative, competitive enzyme linked immunoassay. Concentrations of IaIp measured in pediatric patients suffering from severe DENV infections were significantly lower than in healthy controls.

Conclusions

This is the first report to demonstrate changes in concentration of IaIp during viral infections. The data also highlight the potential of IaIp as a biological marker for severity of DENV infections.     

Noninvasive bioluminescence imaging of dengue virus infection in the brain of A129 mice

Dengue virus (DENV) infection is one of the most important public health threats globally; however, no vaccines or effective antivirals are currently available. The bioluminescence imaging technique has emerged as a powerful tool for studies on viral pathogenesis in vitro and in vivo. In this study, using a recombinant DENV that stably expressed Renilla luciferase (Rluc-DENV), we used bioluminescence for imaging of DENV infection in the brain of A129 mice that lacked type I interferon receptors. Upon intracranial inoculation with Rluc-DENV, A129 mice developed typical neurological symptoms and rapidly succumbed to viral infection. Real-time bioluminescence intensity analysis revealed the replication kinetics of Rluc-DENV in the brain of A129 mice. Linear regression analyses showed a good correlation between photon flux and viral titers (R ²?=?0.9923). Finally, the bioluminescence model was validated using a known mouse monoclonal antibody, 2A10G6, and the therapeutic effects of this neutralizing antibody were readily monitored by live imaging in the same animal. The noninvasive bioluminescence imaging of DENV infection as described here shows distinct advantages over traditional animal models and provides a powerful tool for potential antiviral or vaccine assays against DENV infection in vivo.     

Dengue and Zika viruses: lessons learned from the similarities between these <Emphasis Type="Italic">Aedes</Emphasis> mosquito-vectored arboviruses

The currently spreading arbovirus epidemic is having a severe impact on human health worldwide. The two most common flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), are transmitted through the same viral vector, Aedes spp. mosquitoes. Since the discovery of DENV in 1943, this virus has been reported to cause around 390 million human infections per year, approximately 500,000 of which require hospitalization and over 20,000 of which are lethal. The present DENV epidemic is primarily concentrated in Southeast Asia. ZIKV, which was discovered in 1952, is another important arthropod-borne flavivirus. The neurotropic role of ZIKV has been reported in infected newborns with microcephaly and in adults with Guillain-Barre syndrome. Despite DENV and ZIKV sharing the same viral vector, their complex pathogenic natures are poorly understood, and the infections they cause do not have specific treatments or effective vaccines. Therefore, this review will describe what is currently known about the clinical characteristics, pathogenesis mechanisms, and transmission of these two viruses. Better understanding of the interrelationships between DENV and ZIKV will provide a useful perspective for developing an effective strategy for controlling both viruses in the future.     

Dengue and Zika virus multi-epitope antigen expression in insect cells

Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients’ sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.

     

Dengue Virus in Sub-tropical Northern and Central Viet Nam: Population Immunity and Climate Shape Patterns of Viral Invasion and Maintenance

Dengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV) enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E) gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission.     

Molecular characterization of dengue and chikungunya virus strains circulating in New Delhi,India

Dengue and chikungunya are acute viral infections with overlapping clinical symptoms. Both diseases are transmitted by common mosquito vectors resulting in their co‐circulation in a region. Molecular and serological tests specific for both dengue and chikungunya infections were performed on 87 acute phase blood samples collected from patients with suspected dengue/chikungunya infections in Delhi from September to December, 2011. RT‐PCR and IgM ELISA were performed to detect dengue virus (DENV) and chikungunya virus (CHIKV). NS1 and IgG ELISA were also performed to detect DENV specific antigen and secondary DENV infection. DENV infection was detected in 49%, CHIKV infection in 29% and co‐infection with DENV and CHIKV in 10% of the samples by RT‐PCR. DENV serotypes 1, 2 and 3 were detected in this study. Nine DENV‐1 strains, six DENV‐2 strains and 20 CHIKV strains were characterized by DNA sequencing and phylogenetic analysis of their respective envelope protein genes. DENV‐1 strains grouped in the American African genotype, DENV‐2 strains in the Cosmopolitan genotype and CHIKV strains in the East Central South African genotype by phylogenetic analysis. This is one of the few studies reporting the phylogeny of two dengue virus serotypes (DENV‐1 and DENV‐2) and CHIKV. Surveillance and monitoring of DENV and CHIKV strains are important for design of strategies to control impending epidemics.     

Frequency of FMR1 premutation in individuals with ataxia and/or tremor and/or parkinsonism

A late onset neurological syndrome in carriers of premutation in FMR1 gene was recently described. The condition was named fragile-X-associated tremor/ataxia syndrome (FXTAS) and includes intentional tremor, cerebellar ataxia, parkinsonism, and cognitive deficit. We ascertained the contribution of FMR1 premutation to the phenotypes ataxia, tremor and/or parkinsonism. Sixty-six men over 45 years old presenting these symptoms, isolated or combined, were tested. Also, 74 normal men, randomly chosen in the population, formed the control group. In the patient group, no premutation carrier was found, which is in agreement with other observed frequencies reported elsewhere (0-5% variation). No significant differences were found when comparing gray zone allele frequencies among target and control groups. The FXTAS contribution in patients with phenotypic manifestations of FXTAS was 15/748 (2%). The presence of gray zone alleles is not correlated with FXTAS occurrence.     

Role of mitogen-activated protein kinase signaling in the pathogenesis of dengue virus infection

Dengue virus (DENV) infection is a disease that is endemic to many parts of the world, and its increasing prevalence ranks it among the diseases considered to be a significant threat to public health. The clinical manifestations of DENV infection range from mild dengue fever (DF) to more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased proinflammatory cytokines and vascular permeability, both of which cause organ injury, are the hallmarks of severe dengue disease. Signs of liver injury were observed in studies using hepatic cell lines, mouse models, and autopsy specimens from DENV-infected patients, and these signs substantiated the effects of inflammatory responses and hepatic cell apoptosis. Mitogen-activated protein kinases (MAPK) are involved in inflammatory responses and cellular stress during viral infections. The roles of MAPK signaling in DENV infection were reviewed, and published data indicate MAPK signaling to be involved in inflammatory responses and hepatic cell apoptosis in both in vitro cultures and in vivo models. Modulation of MAPK signaling ameliorates the inflammatory responses and hepatic cell apoptosis in DENV infection. This accumulation of published data relative to the role of MAPK signaling in inflammatory responses and cell apoptosis in DENV infection is elucidatory, and may help to accelerate the development of novel or repositioned therapies to treat this unpredictable and often debilitating disease.     

Sensing of Immature Particles Produced by Dengue Virus Infected Cells Induces an Antiviral Response by Plasmacytoid Dendritic Cells

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.     

Multiple recombinants in two dengue virus, serotype-2 isolates from patients from Oaxaca, Mexico

Background  

Dengue (DEN) is a serious cause of mortality and morbidity in the world including Mexico, where the infection is endemic. One of the states with the highest rate of dengue cases is Oaxaca. The cause of DEN is a positive-sense RNA virus, the dengue virus (DENV) that evolves rapidly increasing its variability due to the absence of a repair mechanism that leads to approximately one mutational event per genome replication; which results in enhancement of viral adaptation, including the escape from host immune responses. Additionally, recombination may play a role in driving the evolution of DENV, which may potentially affect virulence and cause host tropism changes. Recombination in DENV has not been described in Mexican strains, neither has been described the relevance in virus evolution in an endemic state such as Oaxaca where the four serotypes of DENV are circulating.     

Detection of Serotype-Specific Antibodies to the Four Dengue Viruses Using an Immune Complex Binding (ICB) ELISA

Background

Dengue virus (DENV) infections are preferentially diagnosed by detection of specific IgM antibodies, DENV NS1 antigen assays or by amplification of viral RNA in serum samples of the patients. The type-specific immunity to the four worldwide circulating DENV serotypes can be determined by neutralization assays. An alternative to the complicated neutralization assays would be helpful to study the serotype-specific immune response in people in DENV hyperendemic areas but also in subjects upon DENV vaccination.

Methods

In consecutive samples of patients with DENV-1- 4 infection type-specific antibodies were detected using an immune complex binding (ICB) ELISA. During incubation of serum samples and enzyme- labeled recombinant envelope domain III (EDIII) antigens immune complexes (ICs) are formed, which are simultaneously bound to a solid phase coated with an Fc–receptor (CD32). After a single washing procedure the bound labeled ICs can be determined. To further improve type-specific reactions high concentrations of competing heterologous unlabeled ED III proteins were added to the labeled antigens.

Results

Follow-up serum samples of 64 patients with RT-PCR confirmed primary DENV-1, -2, -3 or -4 infections were tested against four enzyme-labeled recombinant DENV EDIII antigens. Antibodies to the EDIII antigens were found in 55 patients (sensitivity 86%). A complete agreement between the serotype detected by PCR in early samples and the serotype-specific antibody in later samples was found. Type-specific anti-EDIII antibodies were first detected 9–20 days after onset of the disease. In 21% of the samples collected from people in Vietnam secondary infections with antibodies to two serotypes could be identified.

Conclusions

The data obtained with the ICB-ELISA show that after primary DENV infection the corresponding type-specific antibodies are detected in almost all samples collected at least two weeks after onset of the disease. The method will be of value to determine the distribution of the various type-specific anti–DENV antibodies in DENV endemic areas.     

The Nicaraguan Pediatric Dengue Cohort Study: Incidence of Inapparent and Symptomatic Dengue Virus Infections, 2004–2010

Dengue, caused by the four serotypes of dengue virus (DENV), is the most prevalent mosquito-borne viral disease of humans. To examine the incidence and transmission of dengue, the authors performed a prospective community-based cohort study in 5,545 children aged 2–14 years in Managua, Nicaragua, between 2004 and 2010. Children were provided with medical care through study physicians who systematically recorded medical consult data, and yearly blood samples were collected to evaluate DENV infection incidence. The incidence of dengue cases observed was 16.1 cases (range 3.4–43.5) per 1,000 person-years (95% CI: 14.5, 17.8), and a pattern of high dengue case incidence every other year was observed. The incidence of DENV infections was 90.2 infections (range 45.2–105.3) per 1,000 person-years (95% CI: 86.1, 94.5). The majority of DENV infections in young children (<6 years old) were primary (60%) and the majority of infections in older children (≥9 years of age) were secondary (82%), as expected. The incidence rate of second DENV infections (121.3 per 1,000 person-years; 95% CI: 102.7, 143.4) was significantly higher than the incidence rate of primary DENV infections (78.8 per 1,000 person-years; 95% CI: 73.2, 84.9). The rigorous analytic methodology used in this study, including incidence reporting in person-years, allows comparison across studies and across different infectious diseases. This study provides important information for understanding dengue epidemiology and informing dengue vaccine policy.     

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.     

Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes – NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.     

Correlation between Dengue-Specific Neutralizing Antibodies and Serum Avidity in Primary and Secondary Dengue Virus 3 Natural Infections in Humans

Although heterotypic secondary infection with dengue virus (DENV) is associated with severe disease, the majority of secondary infections are mild or asymptomatic. The mechanisms of antibody-mediated protection are poorly understood. In 2010, 108 DENV3-positive cases were enrolled in a pediatric hospital-based study in Managua, Nicaragua, with 61 primary and 47 secondary infections. We analyzed DENV-specific neutralization titers (NT₅₀), IgM and IgG avidity, and antibody titer in serum samples collected during acute and convalescent phases and 3, 6, and 18 months post-infection. NT₅₀ titers peaked at convalescence and decreased thereafter. IgG avidity to DENV3 significantly increased between convalescent and 3-month time-points in primary DENV infections and between the acute and convalescent phase in secondary DENV infections. While avidity to DENV2, a likely previous infecting serotype, was initially higher than avidity to DENV3 in secondary DENV infections, the opposite relation was observed 3–18 months post-infection. We found significant correlations between IgM avidity and NT₅₀ in acute primary cases and between IgG avidity and NT₅₀ in secondary DENV infections. In summary, our findings indicate that IgM antibodies likely play a role in early control of DENV infections. IgG serum avidity to DENV, analyzed for the first time in longitudinal samples, switches from targeting mainly cross-reactive serotype(s) to the current infecting serotype over time. Finally, serum avidity correlates with neutralization capacity.     

Dengue virus 3 clinical isolates show different patterns of virulence in experimental mice infection

Dengue virus (DENV) may cause symptomatic infection with mild, undifferentiated febrile illness called classical dengue fever (DF) or a more severe disease, potentially fatal, known as dengue hemorrhagic fever (DHF) or dengue shock syndrome. The pathogenesis of DHF is based on the virulence of the infecting DENV and depends on the infecting serotypes and genotypes; it is also based on the immunopathogenesis that is mediated by host immune responses, including dengue virus-cross-reactive antibodies that augment the severity of infections. Involvement of central nervous system (CNS) is extensively described. The present study describes the virulence of DENV-3 isolates in a mouse model by intracranial (i.c.) inoculation with genotypes I and III. Our data suggest that, in this experimental model, DENV-3 genotype I may have the propensity to cause neurological disease in mice, whereas the genotype III is associated with asymptomatic infection in mice. Additionally, the symptomatic mice show a decrease of white blood cell count, infectious DENV in the brains and alterations in levels of IFN-gamma, IL-6 and MCP-1. The results confirm the mouse model as a way to study the biology of DENV-3 isolates and to improve the knowledge about the neurovirulence of the different genotypes of DENV.