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Recently, in Pakistan, several cases of mysterious dengue‐like illness are being reported, which has concerned the authorities and requires prompt action. We present a case of a 52‐year‐old female patient presenting with a hist...
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Recently, in Pakistan, several cases of mysterious dengue‐like illness are being reported, which has concerned the authorities and requires prompt action. We present a case of a 52‐year‐old female patient presenting with a history of continuous fever, documented up to 104?F, for 5?days. The symptoms were associated with headache, nausea, retro‐orbital headache, arthralgia, and myalgia. Currently, to the best of our knowledge, this is the first reported case in the literature for the endemic mysterious virus and may serve as the groundwork for future studies.
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Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) cons...
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Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (>= 6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties.
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The unexpectedly low vaccine efficacy of Dengvaxia (R), developed by Sanofi Pasteur, and a higher risk of severe diseases after vaccination among dengue-naive children or children younger than 6 years old, have cast skepticism abo...
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The unexpectedly low vaccine efficacy of Dengvaxia (R), developed by Sanofi Pasteur, and a higher risk of severe diseases after vaccination among dengue-naive children or children younger than 6 years old, have cast skepticism about the safety of dengue vaccination resulting in the suspension of school-based immunization programs in the Philippines. The absence of immune correlates of protection from dengue virus (DENV) infection hampers the development of other potential DENV vaccines. While tetravalent live-attenuated tetravalent vaccines (LATVs), which mimic natural infection by inducing both cellular and humoral immune responses, are still currently favored, developing a vaccine that provides a balanced immunity to all four DENV serotypes remains a challenge. With the recently advanced understanding of virion structure and B cell immune responses from naturally infected DENV patients, two points of view in developing a next-generation dengue vaccine emerged: one is to induce potent, type-specific neutralizing antibodies (NtAbs) recognizing quaternary structure-dependent epitopes by having four components of vaccine strains replicate equivalently; the other is to induce protective and broadly NtAbs against the four serotypes of DENV with a universal vaccine. This article reviews the studies related to these issues and the current knowledge gap that needs to be filled in.
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Dengue is an arboviral disease, which threatens almost half the global population, and has emerged as the most significant of current global public health challenges. In this study, we prepared dengue virus-like particles (DENV-LP...
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Dengue is an arboviral disease, which threatens almost half the global population, and has emerged as the most significant of current global public health challenges. In this study, we prepared dengue virus-like particles (DENV-LPs) consisting of Capsid-premembrane-envelope (CprME) and premembrane-envelope (prME) polypeptides from serotype 1 and 4, which were expressed in the silkworms using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid. 1CprME, 1prME, 4CprME, and 4prME expressed proteins in hemolymph, and the molecular weight of the purified proteins was 55?kDa, respectively. The purified polypeptides formed spherical Dengue virus-like particles (DENV-LPs) with ~ 30–55?nm in diameter. The immunoelectron microscopy (IEM) images revealed antigens to the surface of a lipid bilayer of DENV-LPs. The heparin-binding assay shows a positive relationship between absorbance and E protein domain III (EDIII) quantity, which is supported by the isothermal titration calorimetry assay. This indicates a moderate binding affinity between heparin and DENV-LP. The high correlation between patient sera and DENV-LP reactivities revealed that these DENV-LPs shared similar epitopes with the natural dengue virus. IgG elicitation studies in mice have demonstrated that DENV-LPs/CPrMEs elicit a stronger immune response than DENV-LP/prMEs, which lends credence to this claim. Supplementary Information The online version contains supplementary material available at 10.1186/s13568-022-01353-6. Highlights Dengue virus-like particles for serotype 1 and serotype 4 (DENV-LPs/1 and DENV-LPs/4) were produced in silkworm. Heparin-binding assay by ELISA and ITC showed that DENV-LPs/1 and DENV-LPs/4 contain Envelope Domain III. DENV-LPs/1 and DENV-LPs/4 showed affinity to sera from human dengue patients and immunized mice. Supplementary Information The online version contains supplementary material available at 10.1186/s13568-022-01353-6.
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Recent phase Ilb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to deve...
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Recent phase Ilb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested. (C) 2015 Elsevier Ltd. All rights reserved.
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Virus-like particles (VLPs) are shell-like viruses that lack virus-specific genetic materials. Many viral-structured proteins can assemble into VLPs, which mimic the overall structure of virus particles and can elicit strong immun...
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Virus-like particles (VLPs) are shell-like viruses that lack virus-specific genetic materials. Many viral-structured proteins can assemble into VLPs, which mimic the overall structure of virus particles and can elicit strong immune responses in a host. Dengue viruses (DENVs), from the genus Flavivirus, are transmitted to humans through the bites of an infected Aedes mosquito. DENVs cause several diseases that prevailed mainly in tropical and subtropical areas. However, effective treatment measures and preventive strategies for dengue diseases are still lacking. The present minireview summarized the assembly and maturation of DENVs, the strategies and effective factors for dengue VLP construction, and the application of DENV VLPs.
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Objective: Currently, dengue represents one of the most significant arboviral disease worldwide, for which a vaccine is not yet available. Persistent challenges in live viral dengue vaccines have sparked a keen interest in explori...
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Objective: Currently, dengue represents one of the most significant arboviral disease worldwide, for which a vaccine is not yet available. Persistent challenges in live viral dengue vaccines have sparked a keen interest in exploring non-replicating dengue vaccines. We have examined the feasibility of using the methylotrophic yeast Pichia pastoris to develop a chimeric vaccine candidate displaying the dengue virus type-2 (DENV-2) envelope domain III (EDIII), implicated in host receptor binding and in the induction of virus-neutralizing antibodies, on the surface of non-infectious virus-like particles (VLP)-based on the Hepatitis B virus core antigen (HBcAg). Methods: We designed a fusion antigen by inserting DENV-2 EDIII into c/e1 loop of HBcAg. A codon-optimized gene encoding this fusion antigen was integrated into the genome of P. pastoris, under the control of the Alcohol Oxidase 1 promoter. The antigen was expressed by methanol induction and purified to near homogeneity by Ni2+ affinity chromatography. The purified antigen was characterized physically and functionally to evaluate its ability to assemble into VLPs, and elicit DENV-2-specific antibodies in mice. Results: This fusion antigen was expressed successfully to high yields and purified to near homogeneity. Electron microscopy and competitive ELISA analyses showed that it formed VLPs in which the EDIII moiety was accessible to different EDIII-specific antibodies. These VLPs were immunogenic in mice, stimulating the production of antibodies that could specifically recognize DENV-2 and neutralize its infectivity. However, virus-neutralizing antibody titers were modest. Conclusions: Our data show: (i) insertion of EDIII into the c/e1 loop of HBcAg does not compromise particle assembly; and (ii) the chimeric VLPs elicit a specific humoral response against DENV-2. The strategy of displaying dengue virus EDIII using a VLP platform will need further optimization before it may be developed into a viable alternative option.
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Despite the recent introduction of a commercial vaccine, the mosquito-transmitted dengue virus is still a worldwide public health problem. Based on the live attenuated vaccine strategy, the commercial vaccine has a less than optim...
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Despite the recent introduction of a commercial vaccine, the mosquito-transmitted dengue virus is still a worldwide public health problem. Based on the live attenuated vaccine strategy, the commercial vaccine has a less than optimal protective profile. Virus-like particles (VLPs) offer an attractive alternate vaccination strategy due to the effectively native presentation of epitopes in the absence of any infectious genetic material. However, the production of amounts of VLP in a platform that can support commercial development remains a major obstacle. This study generated two DENV 2 VLPs [codon-optimized and chimeric DENV/Japanese encephalitis virus (JEV)] and directly compared yields of these constructs by western blotting and dot blot hybridization. The effect of oleic acid supplementation, a process known to increase DENV production in natural infection, was also investigated. Results showed that the chimeric construct gave a two-to threefold higher yield than the codon-optimized construct and that while oleic acid increased DENV virion production in natural infection, it inhibited VLP production. These results suggest that further optimization of DENV VLP expression is possible, but it will require more understanding of how native DENV infection remodels the host cell machinery.
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Viral hemorrhagic fevers are becoming increasingly common in the tropics and subtropics. Dengue fever is currently the most important arthropod?borne viral disease because of its widespread distribution in more than 100 countries ...
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Viral hemorrhagic fevers are becoming increasingly common in the tropics and subtropics. Dengue fever is currently the most important arthropod?borne viral disease because of its widespread distribution in more than 100 countries and its potential for extensive outbreaks of life?threatening disease. Material and Methods: This study was a hospital?based cross?sectional study conducted in the Microbiology Laboratory of Maternal and Child Tertiary Care Hospital in Western Rajasthan, India, between January 2021 and December 2021. Institutional Ethical Committee permission was obtained. All patients with clinical suspicion of dengue?like illness (DLI), attending outpatient department (OPD) or inpatient department (IPD), were included in the study after obtaining their written consent. A blood sample was collected, and the Dengue Duo rapid card test was conducted for the detection of nonstructural protein 1 (NS1) antigen and immunoglobulin (Ig) M or IgG antibody estimation. All positive samples were tested for IgM enzyme?linked immunosorbent assay (ELISA) test using MAC?ELISA. Results: Of 250 positive sample, the distribution of cases as per clinical features was as follows: all cases presented with fever (100%) followed by myalgia (24.5%), headache (16.06%), hemorrhagic manifestation (13.25%), rash (8.84%), and bleeding gums (2.01%). Thrombocytopenia was seen in 30.40% (76/250) of dengue fever cases. NS1 antigen was detected in 157 cases (62.80%) followed by IgG in 84 cases (33.60%), IgM in 77 cases (30.80%), NS1+IgG in 27 cases (10.80%), NS1 + IgM in 16 cases (6.40%), and NS1 + IgM + IgG in five cases (2%). Of 250 samples, 77 cases were IgM positive and 173 were IgM negative by the Dengue Duo card test. Among the 173 Dengue Duo IgM card negative, 131 cases (79.39%) were also detected negative by IgM ELISA and 42 cases (49.41%) were detected positive by IgM ELISA. The sensitivity was 50.59%, the specificity was 79.39%, the positive predictive value (PPV) was 55.84%, the negative predictive value (NPV) was 75.72%, and the diagnostic accuracy was 69.90%. The case fatality of the cases was 2.35%. Conclusion: Early diagnosis and treatment can prevent mortality in pediatric and pregnant females suffering from dengue and dengue?like illness. Facility and availability of ELISA kits should be adequate for early confirmation of suspected dengue patients by ELISA test.
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Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but lar...
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Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM + E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies. (C) 2014 Elsevier B.V. All rights reserved.
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