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Dengue virus co-circulates as four serotypes, and sequential infections with more than one serotype are common. One hypothesis for the increased severity seen in secondary infections is antibody-dependent enhancement (ADE) leading to increased replication in Fc receptor-bearing cells. In this study, we have generated a panel of human monoclonal antibodies to dengue virus. Antibodies to the structural precursor-membrane protein (prM) form a major component of the response. These antibodies are highly cross-reactive among the dengue virus serotypes and, even at high concentrations, do not neutralize infection but potently promote ADE. We propose that the partial cleavage of prM from the viral surface reduces the density of antigen available for viral neutralization, leaving dengue viruses susceptible to ADE by antibody to prM, a finding that has implications for future vaccine design.

Viruses manipulate the life cycle in host cells via the use of viral properties and host machineries. Development of antiviral peptides against dengue virus (DENV) infection has previously been concentrated on blocking the actions of viral structural proteins and enzymes in virus entry and viral RNA processing in host cells. In this study, we proposed DENV NS1, which is a multifunctional non-structural protein indispensable for virus production, as a new target for inhibition of DENV infection by specific peptides. We performed biopanning assays using a phage-displayed peptide library and identified 11 different sequences of 12-mer peptides binding to DENV NS1. In silico analyses of peptide-protein interactions revealed 4 peptides most likely to bind to DENV NS1 at specific positions and their association was analysed by surface plasmon resonance. Treatment of Huh7 cells with these 4 peptides conjugated with N-terminal fluorescent tag and C-terminal cell penetrating tag at varying time-of-addition post-DENV infection could inhibit the production of DENV-2 in a time- and dose-dependent manner. The inhibitory effects of the peptides were also observed in other virus serotypes (DENV-1 and DENV-4), but not in DENV-3. These findings indicate the potential application of peptides targeting DENV NS1 as antiviral agents against DENV infection.

Dengue infections are increasing at an alarming rate in many tropical and subtropical countries, where epidemics can put health care systems under extreme pressure. The more severe infections lead to dengue hemorrhagic fever (DHF), which can be life threatening. A variety of viral and host factors have been associated with the severity of dengue infections. Because secondary dengue infection is more commonly associated with DHF than primary infections, the acquired immune response to dengue, both B cells and T cells have been implicated. In this study, we set out to study T-cell responses across the entire dengue virus proteome and to see whether these were related to disease severity in a cohort of dengue-infected children from Thailand. Robust responses were observed in most infected individuals against most viral proteins. Responses to NS3 were the most frequent, and there was a very strong association between the magnitude of the response and disease severity. Furthermore, in DHF, cytokine-high CD107a-negative cells predominated.

The 4 dengue virus serotypes (DENV1-4) and Zika virus (ZIKV) are related mosquito-borne flaviviruses of major importance globally. While monoclonal antibodies and plasma from DENV-immune donors can neutralize or enhance ZIKV in vitro and in small-animal models, and vice versa, the extent, duration, and significance of cross-reactivity in humans remains unknown, particularly in flavivirus-endemic regions. We studied neutralizing antibodies to ZIKV and DENV1-4 in longitudinal serologic specimens collected through 3 years after infection from people in Latin America and Asia with laboratory-confirmed DENV infections. We also evaluated neutralizing antibodies to ZIKV and DENV1-4 in patients with Zika through 6 months after infection. In patients with Zika, the highest neutralizing antibody titers were to ZIKV, with low-level cross-reactivity to DENV1-4 that was greater in DENV-immune individuals. We found that, in primary and secondary DENV infections, neutralizing antibody titers to ZIKV were markedly lower than to the infecting DENV and heterologous DENV serotypes. Cross-neutralization was greatest in early convalescence, then ZIKV neutralization decreased, remaining at low levels over time. Patterns of antibody cross-neutralization suggest that ZIKV lies outside the DENV serocomplex. Neutralizing antibody titers can distinguish ZIKV from DENV infections when all viruses are analyzed simultaneously. These findings have implications for understanding natural immunity and vaccines.

[This corrects the article DOI: 10.1371/journal.pone.0327360.].

Dengue virus (DENV) infection is a major global health problem. While DENV infection rarely results in serious complications, the more severe illness dengue hemorrhagic fever (DHF) has a significant mortality rate due to the associated plasma leakage that may lead to hypovolemic shock. Proper care thus requires identifying patients with DHF among those with suspected dengue so that they can be provided with adequate and prompt fluid replacement. In this study we used seventeen years of pediatric patient data from a prospective cohort study in two hospitals in Thailand to develop models to predict DHF among patients with suspected dengue infection. We produced models for a general hospital setting and for a primary care unit setting lacking lab facilities. The best model using combined data from both hospitals achieved an AUC of 0.90 for the general hospital setting and 0.79 for the primary care unit setting. We then investigated the generalizability of the models by training models with data from one hospital and testing them with data from the other. For some models, we found a significant reduction in performance. Possible sources of this are differences in how attributes are defined or measured and differences in the hematological parameters of the two patient populations. We conclude that while high accuracy prediction of DHF is possible, care must be taken when applying DHF predictive models from one clinical setting to another.

Dengue virus (DENV) infection is a significant global health problem. There are no specific therapeutics or widely available vaccines. Early diagnosis is critical for patient management. Viral RNA detection by multiplex RT-PCR using multiple pairs of primers/probes allowing the simultaneous detection of all four DENV serotypes is commonly used. However, increasing the number of primers in the RT-PCR reaction reduces the sensitivity of detection due to the increased possibility of primer dimer formation. Here, a one tube, singleplex real-time RT-PCR specific to DENV 3′-UTR was developed for the detection and quantification of pan-DENV with no cross reactivity to other flaviviruses. The sensitivity of DENV detection was as high as 96.9% in clinical specimens collected at the first day of hospitalization. Our assay provided equivalent PCR efficiency and RNA quantification among each DENV serotype. The assay’s performance was comparable with previously established real-time RT-PCR targeting coding sequences. Using both assays on the same specimens, our results indicate the presence of defective virus particles in the circulation of patients infected with all serotypes. Dual regions targeting RT-PCR enhanced the sensitivity of viral genome detection especially during the late acute phase when viremia rapidly decline and an incomplete viral genome was clinically evident.

The four divergent serotypes of dengue virus are the causative agents of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. About two-fifths of the world's population live in areas where dengue is prevalent, and thousands of deaths are caused by the viruses every year. Dengue virus is transmitted from one person to another primarily by the yellow fever mosquito, Aedes aegypti. Recent studies have begun to define how the dengue viral proteins interact with host proteins to mediate viral replication and pathogenesis. A combined analysis of these studies, however, suggests that many virus-host protein interactions remain to be identified, especially for the mosquito host. In this study, we used high-throughput yeast two-hybrid screening to identify mosquito and human proteins that physically interact with dengue proteins. We tested each identified host protein against the proteins from all four serotypes of dengue to identify interactions that are conserved across serotypes. We further confirmed many of the interactions using co-affinity purification assays. As in other large-scale screens, we identified some previously detected interactions and many new ones, moving us closer to a complete host - dengue protein interactome. To help summarize and prioritize the data for further study, we combined our interactions with other published data and identified a subset of the host-dengue interactions that are now supported by multiple forms of evidence. These data should be useful for understanding the interplay between dengue and its hosts and may provide candidates for drug targets and vector control strategies.

Non-structural protein 1 (NS1) is a glycoprotein component of dengue virus (DENV) that is essential for viral replication, infection and immune evasion. Immunization with NS1 has been shown to elicit antibody-mediated immune responses which protect mice against DENV infections. Here, we obtained peripheral blood mononuclear cells from human subjects with secondary dengue infections, which were used to construct a dengue immune phage library displaying single-chain variable fragments. Phage selective for DENV NS1 were obtained by biopanning. Twenty-one monoclonal antibodies (mAbs) against DENV NS1 were generated from the selected phage and characterized in detail. We found most anti-NS1 mAbs used IGHV1 heavy chain antibody genes. The mAbs were classified into strongly and weakly-reactive groups based on their binding to NS1 expressed in dengue virus 2 (DENV2)-infected cells. Antibody binding experiments with recombinant NS1 proteins revealed that the mAbs recognize conformational epitopes on the β-ladder domain (amino acid residues 178–273) of DENV NS1. Epitope mapping studies on alanine-substituted NS1 proteins identified distinct but overlapping epitopes. Protruding amino acids distributed around the spaghetti loop are required for the binding of the strongly-reactive mAbs, whereas the recognition residues of the weakly-reactive mAbs are likely to be located in inaccessible sites facing toward the cell membrane. This information could guide the design of an NS1 epitope-based vaccine that targets cross-reactive conserved epitopes on cell surface-associated DENV NS1.

Dengue virus is a major pathogen, and severe infections can lead to life-threatening dengue hemorrhagic fever. Dengue virus exists as four serotypes, and dengue hemorrhagic fever is often associated with secondary heterologous infections. Antibody-dependent enhancement (ADE) may drive higher viral loads in these secondary infections and is purported to result from antibodies that recognize dengue virus but fail to fully neutralize it. Here we characterize two antibodies, 2C8 and 3H5, that bind to the envelope protein. Antibody 3H5 is highly unusual as it not only is potently neutralizing but also promotes little if any ADE, whereas antibody 2C8 has strong capacity to promote ADE. We show that 3H5 shows resilient binding in endosomal pH conditions and neutralizes at low occupancy. Immunocomplexes of 3H5 and dengue virus do not efficiently interact with Fcγ receptors, which we propose is due to the binding mode of 3H5 and constitutes the primary mechanism of how ADE is avoided. Immunity to one serotype of dengue virus can worsen disease following exposure to another serotype, a process called ‘antibody-dependent enhancement’. Grimes and colleagues characterize the function and structural basis of an unusual, potent and broadly neutralizing antibody that lacks such activity.