Dengue Virus NS1 Research Articles

A T164S mutation in the dengue virus NS1 protein is associated with greater disease severity in mice.

Dengue viruses cause severe and sudden human epidemics worldwide. The secreted form of the nonstructural protein 1 (sNS1) of dengue virus causes vascular leakage, a hallmark of severe dengue disease. Here, we reverse engineered the T164S mutation of NS1, associated with the severity of dengue epidemics in the Americas, into a dengue virus serotype 2 mildly infectious strain. The T164S mutant virus decreased infectious virus production and increased sNS1 production in mammalian cell lines and human peripheral blood mononuclear cells (PBMCs) without affecting viral RNA replication. Gene expression profiling of 268 inflammation-associated human genes revealed up-regulation of genes induced in response to vascular leakage. Infection of the mosquito vector Aedes aegypti with the T164S mutant virus resulted in increased viral load in the mosquito midgut and higher sNS1 production compared to wild-type virus infection. Infection of type 1 and 2 interferon receptor-deficient AG129 mice with the T164S mutant virus resulted in severe disease coupled with increased complement activation, tissue inflammation, and more rapid mortality compared to AG129 mice infected with wild-type virus. Molecular dynamics simulations predicted that mutant sNS1 formed stable dimers similar to the wild-type protein, whereas the hexameric mutant sNS1 was predicted to be unstable. Immunoaffinity-purified sNS1 from T164S mutant virus-infected mammalian cells was associated with different lipid classes compared to wild-type sNS1. Treatment of human PBMCs with sNS1 purified from T164S mutant virus resulted in a twofold higher production of proinflammatory cytokines, suggesting a mechanism for how mutant sNS1 may cause more severe dengue disease.

Development of an Enzyme-Linked Immunosorbent Assay for Rapid Detection of Dengue Virus (DENV) NS1 and Differentiation of DENV Serotypes during Early Infection.

Dengue fever, caused by infections with the dengue virus (DENV), affects nearly 400 million people globally every year. Early diagnosis and management can reduce the morbidity and mortality rates of severe forms of dengue disease as well as decrease the risk of wider outbreaks. Although the early diagnosis of dengue can be achieved using a number of commercial NS1 detection kits, none of these can differentiate among the four dengue virus serotypes. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for the detection of dengue virus (DENV) NS1 by pairing a serotype-cross-reactive monoclonal antibody (MAb) with one of four serotype-specific MAbs in order to facilitate the rapid detection of NS1 antigens and the simultaneous differentiation of DENV serotypes. A total of 146 serum samples obtained from patients suspected to be in the acute phase of DENV infection were used to evaluate the clinical application of our novel test for the detection and serotyping of DENV. The overall sensitivity rate of our test was 84.85%, and the sensitivity rates for serotyping were as follows: 88.2% (15/17) for DENV serotype 1 (DENV1), 94.7% (18/19) for DENV2, 75% (12/16) for DENV3, and 66.6% (6/9) for DENV4. Moreover, there was no cross-reactivity among serotypes, and no cross-reactivity was observed in sera from nondengue patients. Thus, our test not only enables the rapid detection of the dengue virus but also can distinguish among the specific serotypes during the early stages of infection. These results indicate that our ELISA for DENV NS1 is a convenient tool that may help elucidate the epidemiology of DENV outbreaks and facilitate the clinical management of DENV infections.

STAT2 is a determinant of yellow fever virus host tropism

Abstract The recent yellow fever virus (YFV) epidemic in Brazil in 2017 and Zika virus (ZIKV) epidemic in 2015 serve to remind us of the importance of flaviviruses as emerging human pathogens. With the current global flavivirus threat, there is an urgent need for antivirals and vaccines to curb the spread of these viruses. However, the lack of suitable small animal models limits the research questions that can be answered. A common trait of all flaviviruses studied thus far is their ability to antagonize interferon (IFN) signaling so as to establish infection and subsequent dissemination. Previously, we reported that YFV NS5 requires the presence of type I IFN (IFNα/β) for its engagement with human Signal Transducer and Activator of Transcription 2 (hSTAT2). We now report that like the NS5 proteins of ZIKV and dengue virus (DENV), YFV NS5 protein is able to bind hSTAT2 but not murine STAT2 (mSTAT2). Contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, replacing mSTAT2 with hSTAT2 cannot rescue the YFV NS5-STAT2 interaction, as YFV NS5 is also unable to interact with hSTAT2 in murine cells. We show that the IFNα/β-dependent ubiquitination of YFV NS5 that is required for STAT2 binding in human cells is absent in murine cells. In addition, we demonstrate that mSTAT2 restricts YFV replication in vivo. These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses.

Smartphone-based fluorescent lateral flow immunoassay platform for highly sensitive point-of-care detection of Zika virus nonstructural protein 1

Simple, inexpensive, and rapid diagnostic tests in low-resource settings with limited laboratory equipment and technical expertise are instrumental in reducing morbidity and mortality from epidemic infectious diseases. We developed a smartphone-based fluorescent lateral flow immunoassay (LFIA) platform for the highly sensitive point-of-care detection of Zika virus nonstructural protein 1 (ZIKV NS1). An attachment was designed and 3D-printed to integrate the smartphone with external optical and electrical components, enabling the miniaturization of the instrument and reduction in cost and complexity. Quantum dot microspheres were utilized as probes in fluorescent LFIA because of their extremely bright fluorescence signal. This approach can achieve quantitative point-of-care detection of ZIKV NS1 within 20 min. Limits of detection (LODs) in buffer and serum were 0.045 and 0.15 ng mL−1, respectively. Despite the high structural similarity, a high-level Dengue virus NS1 as interferent showed limited cross-reactivity. Furthermore, this assay was successfully applied to detecte ZIKV NS1 and virions spiked in complex biological samples, indicating its practical application capability. Given its low cost, compact size, and excellent analytical performance, the proposed smartphone-based fluorescent LFIA platform holds considerable potential in rapid and accurate point-of-care detection of ZIKV NS1 and provides new insight into the design and application of molecular diagnostic methods in low-resource settings.

An estrogen antagonist, cyclofenil, has anti-dengue-virus activity.

Dengue virus (DENV) infections are a major cause of morbidity and mortality in tropical and subtropical areas. Several compounds that act against DENV have been studied in clinical trials to date; however, there have been no compounds identified that are effective in reducing the severity of the clinical manifestations. To explore anti-DENV drugs, we examined small molecules that interact with DENV NS1 and inhibit DENV replication. Cyclofenil, which is a selective estrogen receptor modulator (SERM) and has been used clinically as an ovulation-inducing drug, showed an inhibitory effect on DENV replication in mammalian cells but not in mosquito cells. Other SERMs also inhibited DENV replication in mammalian cells, but cyclofenil showed the weakest cytotoxicity among these SERMs. Cyclofenil also inhibited the replication of Zika virus. A time-of-addition assay suggested that cyclofenil may interfere with two stages of the DENV life cycle: the translation-RNA synthesis and assembly-maturation stages. However, the level of intracellular infectious particles decreased more drastically after treatment with cyclofenil than the viral RNA level did, indicating that the assembly-maturation stage might be the main target of cyclofenil. In electron microscopy analysis, many aggregated particles were detected in DENV-infected cells in the presence of cyclofenil, supporting the possibility that cyclofenil impedes the process of assembly and maturation of DENV.

Optimization of a fragment linking hit toward Dengue and Zika virus NS5 methyltransferases inhibitors

No antiviral drugs to treat or prevent life-threatening flavivirus infections such as those caused by mosquito-borne Dengue (DENV) and more recently Zika (ZIKV) viruses are yet available. We aim to develop, through a structure-based drug design approach, novel inhibitors targeting the NS5 AdoMet-dependent mRNA methyltransferase (MTase), a viral protein involved in the RNA capping process essential for flaviviruses replication. Herein, we describe the optimization of a hit (5) identified using fragment-based and structure-guided linking techniques, which binds to a proximal site of the AdoMet binding pocket. X-ray crystallographic structures and computational docking were used to guide our optimization process and lead to compounds 30 and 33 (DENV IC50 = 26 μM and 23 μM; ZIKV IC50 = 28 μM and 19 μM, respectively), two representatives of novel non-nucleoside inhibitors of flavivirus MTases.

Validation of serological and molecular methods for diagnosis of zika virus infections

The laboratory confirmation of Zika virus (ZIKV) infection, and the differential diagnosis from other flavivirus infections such as dengue virus (DENV), often requires the use of several diagnostic test types. Cross-reactions and secondary infections complicate the serological diagnosis and specific viral RNA detection assays are often needed for confirming the diagnosis.The aim of this study was to validate serological and molecular methods for diagnosing ZIKV infection. This included the evaluation of a ZIKV RT-qPCR assay for diagnostics that was previously set up for research use and to compare the ZIKV, DENV and TBEV EIA methods. External and in-house controls and pre-characterized sample panels were tested, and also automated and manual nucleic acid extraction methods were compared.A total of ten Finnish traveler patients were diagnosed with acute ZIKV infection during 2015–2017 including one suspected dual DENV and ZIKV infection. These samples along with panels of DENV and tick-borne encephalitis virus (TBEV) infections were used to test the cross-reactive properties of ZIKV, DENV and TBEV IgM assays. Additionally, the diagnosed acute ZIKV patient samples were tested using commercially available diagnostic DENV NS1 antigen assay and a ZIKV NS1 antigen assay intended for research use.The ZIKV RT-qPCR assay was demonstrated to be both specific and sensitive (one genome per reaction) and suitable for routine diagnostic use utilizing automated nucleic acid extraction. Of the tested IgM tests the NS1 antigen-based ZIKV IgM (Euroimmun) assay performed with least cross-reactivity with a specificity of 97.4%. The DENV IgM assay (Focus Diagnostics) had specificity of only 86.1%. The results are in line with previous studies and additionally highlight that also acute TBEV patients may give a false positive test result in DENV and ZIKV IgM assays.

Low frequency of asymptomatic dengue virus-infected donors in blood donor centers during the largest dengue outbreak in Taiwan.

To determine the prevalence of asymptomatic dengue virus-infected blood donors during the largest dengue outbreak in Taiwan history occurred in 2015, we examined the evidence of dengue virus (DENV) infection by the detection of DENV RNA genome using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), DENV NS1 antigen using rapid diagnosis test (RDT) and anti-dengue antibody using IgM/IgG capture enzyme-linked immunosorbent assay (capture ELISA) and RDT in eight thousand serum samples from blood donations to the blood centers of the Taiwan Blood Services Foundation (TBSF) in Kaohsiung City and Tainan City during the largest dengue outbreak in Taiwan history occurred in 2015. Only one serum sample was positive for DENV RNA detection by using dengue-specific real-time RT-PCR, the virus was DENV-2 determined by serotype-specific real-time RT-PCR and sequencing, and the DENVs in the serum were confirmed as being infectious by a plaque assay. The recipient of this blood did not develop any dengue fever symptom on follow-up. None of the samples was NS1 RDT-reactive. Seventeen IgM-positive samples were identified. There was a low prevalence of asymptomatic confirmed or probable DENV-infected blood donors in our study (0.013% and 0.21%, respectively), and no symptomatic transfusion-transmitted dengue (TT dengue) was developed during the largest dengue outbreak in Taiwan history in highly endemic areas and periods.

Cross-Reactivity and Anti-viral Function of Dengue Capsid and NS3-Specific Memory T Cells Toward Zika Virus.

Zika virus (ZIKV), a flavivirus with homology to dengue virus (DENV), is spreading to areas of DENV hyper-endemicity. Heterologous T cell immunity, whereby virus-specific memory T cells are activated by variant peptides derived from a different virus, can lead to enhanced viral clearance or diminished protective immunity and altered immunopathology. In mice, CD8+ T cells specific for DENV provide in vivo protective efficacy against subsequent ZIKV infection. In humans, contrasting studies report complete absence or varying degrees of DENV/ZIKV T cell cross-reactivity. Moreover, the impact of cross-reactive T cell recognition on the anti-viral capacity of T cells remains unclear. Here, we show that DENV-specific memory T cells display robust cross-reactive recognition of ZIKV NS3 ex vivo and after in vitro expansion in respectively n = 7/10 and n = 9/9 dengue-immune individuals tested. In contrast, cross-reactivity toward ZIKV capsid is low or absent. Cross-reactive recognition of DENV or ZIKV NS3 peptides elicits similar production of the anti-viral effector mediators IFN-γ, TNF-α, and CD107a. We identify 9 DENV/ZIKV cross-reactive epitopes, 7 of which are CD4+ and 2 are CD8+ T cell epitopes. We also show that cross-reactive CD4+ and CD8+ T cells targeting novel NS3 epitopes display anti-viral effector potential toward ZIKV-infected cells, with CD8+ T cells mediating direct lyses of these cells. Our results demonstrate that DENV NS3-specific memory T cells display anti-viral effector capacity toward ZIKV, suggesting a potential beneficial effect in humans of pre-existing T cell immunity to DENV upon ZIKV infection.

Bio-Functionalized Tapered Multimode Fiber Coated With Dengue Virus NS1 Glycoprotein for Label Free Detection of Anti-Dengue Virus NS1 IgG Antibody

This proposed paper demonstrates the development of a bio-functionalized tapered multimode optical fiber for the detection of anti-Dengue virus NS1 Immunoglobin G (IgG). The tapered fiber with waist diameter and waist length of 10 $\mu \text{m}$ and 10 mm, respectively, promotes the penetration of light modes to the core-cladding interface, creating evanescent wave that are sensitive to changes on the fiber surface. As the fiber is bio-functionalized with Dengue virus (DENV) NS1 glycoprotein specific to anti-DENV NS1 IgG antibodies, alteration on the surface is selective towards the presence of IgG antibodies. The absorbance-based sensor obtained a sensitivity value of $7 \times 10$ −6 a.u/pg/mL with a detection time under 2 min. This paper shows great potential for real-world deployment which impact may bring Dengue diagnostics a step further.

Cloning and Expression of NS3 Gene of Pakistani Isolate Type 2 Dengue Virus.

IntroductionDengue is one of the major emerging viral diseases in the world, with dramatic increases in reported cases in the last few decades and annual worldwide occurrence of approximately 390 million infections. It is a highly important mosquito-vectored disease and is a problem in tropical and subtropical areas of the world. The major aim of this study was to clone and express the dengue NS3 gene, in service to its therapeutic importance for the development of stable cell lines.Material and MethodsBlood samples from dengue fever (DF) patients were collected and subjected to PCR amplification of the NS3 gene of dengue virus serotype-2 (DENV-2). The NS3 gene was amplified using gene specific primers and cloned in the TA cloning vectors.ResultsThe gene was successfully expressed in mammalian expression vector pcDNA3.1. The current finding was different from a previously reported DENV-2 strain replicon constructed in different cells, in which the whole genetic material of the virus was used instead of an active protease gene, and which gave a low yield of replicon expressing cells.ConclusionRecombinant NS3 could be used to produce an antibody that is possibly helpful for developing a single step diagnostic assay to detect the dengue virus NS3 antigen in sera of dengue patients.

KETERKAITAN ANTIGEN NS1 INFEKSI VIRUS DENGUE DENGAN SEROTIPE VIRUS DENGUE

Dengue virus infection (DVI) currently is detected by using dengue virus NS1 antigen (NS1 Ag). The sensitivity of NS1 Ag is 27.8%–93.4%,but recent study of Kumarasamy the sensitivity of NS1 Ag is better than the virus isolation and polymerase chain reaction (PCR). This studyis focussed on the evaluation of the validity of Panbio Dengue Early Rapid for the diagnosis of DVI and the NS1 Ag sensitivity associated withdengue virus serotypes. The sera was obtained from 65 DVI patients which diagnosed by the clinicians. The resulted diagnosis was foundby serology tests (positive IgM/IgG antidengue/NS1 Ag ELISA) and 1997 WHO criteria as the gold standard, and which also found 35 nonDVI patients (typhoid fever, HAV, malaria, UTI, tuberculosis and bronchopneumonia). The samples were examined by Panbio Dengue EarlyRapid. PCR was performed on each positive serological test result to determine the dengue virus serotypes. The sensitivity and specificity ofPanbio Dengue Early Rapid was 49.2% and 100%. The PCR results of 65 sera showed positive PCR in 49.2% (positive NS1 Ag was 62.5%).Meanwhile, and negative PCR in 50.8% (positive NS1 Ag was 36.4%). The predominance of serotypes (positive NS1 Ag) were DEN-3 (37.5%),DEN-4 (28.1%), DEN-1 (21.9%) and DEN-2 (12.5%). The Panbio Dengue Early Rapid can be used as early detection of DVI, although itshould be used in conjunction with other dengue serological tests as well. Unfortunately there is still not enough evidence about the NS1 Agsensitivity associated with the dengue virus serotypes.

Production of Immunologically Active Untagged Recombinant DENV-2 NS1 in Escherichia coli

Dengue is an infectious disease affecting 390 millions people in tropical and subtropical region annually. Dengue virus NS1 protein plays an important role in viral replication in the host cell and it is detected in high level in the infected patient serum. A synthetic gene of untagged DENV-2 NS1 with codons optimization for expression in Escherichia coli has been generated and then inserted into an expression vector pET16b. As a hydrophobic protein with aliphatic index of 71.21%, DENV-2 NS1 was produced as inclusion bodies in E. coli BL21(DE3). The DENV-2 NS1 aggregate was unfolded in 8 M urea and then refolded by reverse dilution method. The refolded DENV-2 NS1 has immunologically active structure as it is capable to interact with anti-NS1 antibody, hence making it as a potential vaccine candidate.

Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities.

It has been estimated for dengue infection that the global population at risk is 3.5 billion people, which makes dengue an important public health problem. The causative agents of dengue are dengue viruses. For dengue virus replication, the dengue virus NS5 protein is of special importance as it has several enzyme activities important for viral replication. Previous reports of phosphorylation and SUMOylation of dengue NS5 have shown these protein modifications have important consequences for NS5 functions. In this report we identify glutathionylation, another reversible post translation modification that impacts on NS5 enzyme activity. Using dengue virus infected cells we employed specific antibodies and mass spectrometry to identify 3 cysteine residues of NS5 protein as being glutathionylated. Glutathionylation is a post translational protein modification where glutathione is covalently attached to a cysteine residue. We showed glutathionylation occurs on 3 conserved cysteine residues of dengue NS5. Then we generated two flavivirus recombinant full length proteins, dengue NS5 and Zika NS5, to characterize two of the NS5 enzyme activities, namely, guanylyltransferase and RNA-dependent RNA polymerase activities. We show glutathionylation of dengue and Zika NS5 affects enzyme activities of the two flavivirus proteins. The data suggests that glutathionylation is a general feature of the flavivirus NS5 protein and the modification has the potential to modulate several of the NS5 enzyme functions.

Blood group AB is associated with severe forms of dengue virus infection.

Several viral and host factors are believed to contribute to the development of severe forms of dengue such as dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) following a dengue virus (DENV) infection. The pathogenesis of DHF/DSS is not fully understood, however, host factors like ABO blood groups have been shown to contribute to the severity of DENV infection. The present study investigated the association of blood groups with severity of DENV infection in the northern region of Sri Lanka. The blood-groups of 405 patients positive for DENV NS1 antigen and anti-DENV IgM/IgG were determined using the standard haemagglutination assay recommended by the national blood bank/s. The occurrence of severe dengue in patients with certain blood groups was significantly different (p<0.001) to those with other blood groups. Patients with AB blood group had more than 2.5 times higher risk of developing DHF than those with other blood groups. On the other hand, patients with blood group O were significantly under represented for DHF relative to the proportion of this blood group in the general population. Thus dengue patients with blood group O appear to have a low risk of developing DHF than those with other blood groups.

Zika Immunoassay Based on Surface-Enhanced Raman Scattering Nanoprobes.

Zika virus (ZIKV) was considered a public health emergency of international concern after the 2015 outbreak. Serological tests based on immunoassay platforms is one of the methods applied on the diagnosis of ZIKV and Dengue virus (DENV). However, the high limits of detection (LOD) and the cross-reactivity between ZIKV and DENV are still limitations in immunological tests. In order to tackle these issues, we have designed an immune-specific assay based on surface-enhanced Raman scattering (SERS) nanoprobes. Gold shell-isolated nanoparticles (Au-SHINs) were synthesized with 100 nm Au nanoparticles and 4 nm silica shell thickness coated with Nile Blue (Raman reporter). Then, the SERS nanoprobes were wrapped in a final silica shell and functionalized with monoclonal anti-ZIKV NS1 antibodies. Concentrations of ZIKV NS1 down to 10 ng/mL were probed free of cross-reactivity with DENV NS1 antigens.

The Transactions of NS3 and NS5 in Flaviviral RNA Replication.

Dengue virus (DENV) replication occurs in virus-induced vesicles that contain the replication complex (RC) where viral RNA, viral proteins and host proteins participate in RNA-RNA, RNA-protein and protein-protein interactions to ensure viral genome synthesis. However, the details of the multitude of interactions involved in the biogenesis of the infectious virion are not fully understood. In this review, we will focus on the interaction between non-structural (NS) proteins NS3 and NS5, as well as their interactions with viral RNA and briefly also the interaction of NS5 with the host nuclear transport receptor protein importin-α. The multifunctional NS3 protease/helicase and NS5 methyltransferase (MTase)/RNA-dependent RNA polymerase (RdRp) contain all the enzymatic activities required to synthesize the viral RNA genome. The success stories of drug discovery and development with Hepatitis C virus (HCV), a member of the Flaviviridae family, has led to the view that DENV NS3 and NS5 may be attractive antiviral drug targets. However, more than 10years of intensive research effort by Novatis has revealed that they are not "low hanging fruits" and therefore, the search for potent directly acting antivirals (DAAs) remains a pipeline goal for several medium to large drug discovery enterprises. The effort to discover DAAs for DENV has been boosted by the epidemic outbreak of the closely related flavivirus member- Zika virus (ZIKV). Because the viral RNA replication occurs within a molecular machine that is composed several viral and host proteins, much interest has turned to characterising functionally essential protein-protein interactions in order to identify potential allosteric inhibitor binding sites within the RC.

Roles and Prospects of Dengue Virus Non-structural Proteins as Antiviral Targets: An Easy Digest.

Dengue is a neglected disease caused by the infection of dengue virus which is transmitted by Aedes mosquitoes and to some, it could be fatal. Regardless of the enormous work devoted to research for the treatment of dengue, to this day there is no cure, and treatment is solely limited to supportive care by treating the symptoms. The inhibition of the viral RNA non-structural enzymes has been the most popular approach amongst the strategies applied to the search and development of dengue antivirals. This review is a compact digest of what is already known of the roles and the prospects of the dengue virus non-structural proteins NS1, NS2BNS3, NS4A, NS4B and NS5 as the targets for antiviral studies including the recent progress that has been published regarding their roles.

Sensitive detection of dengue virus NS1 by highly stable affibody-functionalized gold nanoparticles

The anti-NS1 affibody-functionalized gold nanoparticles based ELISA resulted in a 14.2-fold signal amplification performance for dengue NS1 detection.

K48-linked polyubiquitination of dengue virus NS1 protein inhibits its interaction with the viral partner NS4B

Dengue virus (DENV) is a member of the Flaviviridae family, which is transmitted to mammalian species through arthropods, and causes dengue fever or severe dengue fever in humans. The DENV genome encodes for multiple nonstructural (NS) proteins including NS1. NS1 plays an essential role in replication by interacting with other viral proteins including NS4B, however how these interactions are regulated during virus infection is not known. By using bioinformatics, mass spectrometry analysis, and co-immunoprecipitation assays, here we show that DENV-NS1 is ubiquitinated on multiples lysine residues during DENV infection, including K189, a lysine residue previously shown to be important for efficient DENV replication. Data from in vitro and cell culture experiments indicate that dengue NS1 undergoes modification with K48-linked polyubiquitin chains, which usually target proteins to the proteasome for degradation. Furthermore, ubiquitinated NS1 was detected in lysates as well as in supernatants of human and mosquito infected cells. Ubiquitin deconjugation of NS1 using the deubiquitinase OTU resulted in increased interaction with the viral protein NS4B suggesting that ubiquitinated NS1 has reduced affinity for NS4B. In support of these data, a K189R mutation on NS1, which abrogates ubiquitination on amino acid residue 189 of NS1, also increased NS1-NS4B interactions. Our work describes a new mechanism of regulation of NS1-NS4B interactions and suggests that ubiquitination of NS1 may affect DENV replication.