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Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand; 5Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 6Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanBackground: Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4) are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and results: To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the envelope and nonstructural 1 proteins. Phylogenetic distances between the four serotypes of DENV were as different as those of other flaviviruses, such as Japanese encephalitis virus and West Nile virus. Large variations in the DENV serotypes were comparable with the differences between species of flavivirus. Furthermore, the diversity of flavivirus capsid protein was much greater than that of envelope and nonstructural 1 proteins.Conclusion: In this study, we produced specific monoclonal antibodies that can be used to detect DENV-2 capsid protein, but not a cross-reactive one with all serotypes of DENV capsid protein. The high diversity of the DENV capsid protein sequence by phylogenetic analysis supported the low cross-reactivity of monoclonal antibodies against DENV capsid protein.Keywords: Dengue virus, capsid protein, monoclonal antibody, cross-reactivity

Plants belonging to the genus Curcuma have shown promise in exerting antiviral activity. In this study, Curcuma sp. "Khamin Oi" (CKO), a traditional Thai medicinal herb listed in Thai herbal pharmacopoeia, was subjected to extraction using a variety of solvents. Consequently, LC-MS/MS and GC-MS/MS analyses were conducted to assess the phytochemical profiles of all the crude extracts and identify the compounds potentially responsible for their antiviral effects. The antiviral effects of these extracts against the human coronavirus OC43 (HCoV-OC43) were also investigated. An in-cell ELISA was used to investigate the anti-HCoV-OC43 replication activity of the crude rhizome extracts of CKO. Among the extracts, the crude hexane extract of CKO exhibited the lowest IC 50 (3.62 μg/mL), with a high selectivity index (SI) of 31.90, despite having the lowest curcuminoid content. While the methanolic extract of Curcuma longa L., known for its high curcuminoid content among Curcuma species, showed an IC 50 of 14.83 μg/mL with an SI of 2.80. Further in vitro and in vivo investigations of CKO crude extract are necessary to understand its potential anti-HCoV-OC43 properties., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Piriyakorn Pichetpongtorn reports a relationship with 10.13039/501100004156Mahidol University that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is pivotal in facilitating viral entry and serves as a major target for vaccine development and therapeutics. Despite undergoing mutations aimed at evading host immunity, certain regions within the RBD remain conserved., Objective: This study aimed to identify peptides capable of interacting with these conserved regions of the RBD across various variants and assess their neutralization potential., Methods: The PhD-12 phage display library underwent screening to identify phages binding to the RBD. Selected phage clones were examined for binding to the RBD of multiple variants, including 2019-nCoV, Delta (B.1.617.2), Omicron (B.1.1.529), and XBB. Peptides, expressed as chimeric constructs, were tested for their binding to the RBD, the Omicron trimeric S, inactivated SARS-CoV-2 virus, and neutralizing activity. The binding sites were analyzed using Molecular Docking., Results: Two selected phage clones displayed peptides binding to the RBD of multiple variants. Chimeric T hioredoxin-peptides (Trx-RB9 and Trx-RB10) exhibited binding to both inactivated SARS-CoV-2 and the Omicron trimeric S, with half-maximum effective concentrations (EC50 ) values of 111.9 and 360.2 nM, respectively. Molecular docking revealed distinct binding sites within the RBD of the Omicron trimeric S for both Trx-RB9 and Trx-RB10. A mixture of Trx-RB9 and Trx-RB10 inhibited 78% of the binding of recombinant human ACE2 to the Omicron trimeric S., Conclusions: The chimeric Trx-RB9 and Trx-RB10 peptides bind to the RBD of SARS-CoV-2 variants and inhibit the binding of ACE2 to the RBD of the Omicron trimeric S.

Hemagglutinin (HA) is the major envelope glycoprotein and antigen on the surface of influenza virions. The glycoprotein comprises a globular head and a stalk region. While immunodominant epitopes on influenza HA head are highly variable, the stalk domain is conserved. The variability of the HA head causes the antigenic drift that made the requirement of annual update of vaccine strains. Induction of antibody against the stalk domain has been proposed as an approach for a broadly protective influenza vaccine strategy. Sequential exposure to influenza strains with highly diverse HA heads but conserved stalks have been shown to induce antibody to the low immunogenic stalk domain. Here, we tested this approach by using old influenza vaccine strains that are decades apart in evolution. Inactivated whole virion vaccine of influenza A/Puerto Rico/8/1934, A/USSR/92/1977, and A/Thailand/102/2009 (H1N1) was sequentially immunized into BALB/c mice in comparison to immunization using single strain (A/Thailand/102/2009 (H1N1)). The sequentially immunized mice developed higher levels of binding antibody to the stalk domain. These suggested that using old vaccine strains in sequential vaccination may be a possible approach to induce antibody to the conserved stalk domain., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2024 Alita Kongchanagul et al.)

In addition to the well-known differences among the four dengue serotypes, intra-serotypic antigenic diversity has been proposed to play a role in viral evolution and epidemic fluctuation. A replacement of genotype II by genotype III of dengue virus serotype 3 (DENV3) occurred in Thailand during 2007-2014, raising questions about the role of intra-serotypic antigenic differences in this genotype shift. We characterized the antigenic difference of DENV3 of genotypes II and III in Thailand, utilizing a neutralizing antibody assay with DENV3 vaccine sera and monotypic DENV3 sera. Although there was significant antigenic diversity among the DENV3, it did not clearly associate with the genotype. Our data therefore do not support the role of intra-serotypic antigenic difference in the genotype replacement. Amino acid alignment showed that eight positions are potentially associated with diversity between distinct antigenic subgroups. Most of these amino acids were found in envelope domain II. Some positions (aa81, aa124, and aa172) were located on the surface of virus particles, probably involving the neutralization sensitivity. Notably, the strains of both genotypes II and III showed clear antigenic differences from the vaccine genotype I strain. Whether this differencewill affect vaccine efficacy requires further studies.

Background: Upper respiratory tract is the primary target of SARS-CoV-2. Therefore, nasal immune responses act as the first line of defense against SARS-CoV-2 infection., Objective: We aim to investigate the immune responses of human nasal epithelial cells (HNEpCs) upon stimulation with a COVID-19 vaccine candidate. This candidate named RBD-NPs is composed of SARS-CoV-2 receptor-binding domain (RBD) encapsulated within the N,N,N-trimethyl chitosan nanoparticles (TMC-NPs)., Methods: HNEpCs were stimulated with RBD-NPs, empty NPs, or soluble RBD at various concentrations. After 24 and 48 h of treatment, cells viability and delivery of the immunogens were assessed using XTT assay and flow cytometry. Levels of cytokines and chemokines in the supernatant were quantified with Bio-plex Human Cytokine Assay. Communication between RBD-NPs-stimulated HNEpCs and monocyte-derived dendritic cells (MoDCs) was assessed through differentiation of MoDCs into mature phenotype., Results: RBD-NPs as high as 100 μg exerted no toxicity to HNEpCs and could effectively be delivered to HNEpCs. Treatment of HNEpCs with RBD-NPs strongly activated production of several pro-inflammatory cytokines, chemokines, Th1-related cytokines and the monocytes/macrophages growth factors. Interestingly, soluble mediators secreted from RBD-NPs treated HNEpCs significantly upregulated the expression of maturation markers (CD80, CD83, CD86 and HLA-DR) on the MoDCs., Conclusion: This study demonstrated that our COVID-19 vaccine candidate drove HNEpCs into immunologically competent cells that not only exerted anti-viral innate immune responses but also potently induced MoDCs maturation.

Cyclophilin A (CypA) or peptidylprolyl isomerase A, plays an important role in protein folding, trafficking, environmental stress, cell signaling and apoptosis etc. In shrimp, the mRNA expression level of PmCypA was stimulated by LPS. In this study, all three types of shrimp hemocytes: hyaline cell, granulocyte and semi-granulocyte expressed the PmCypA protein. The mRNA expression level of PmCypA was found to be up-regulate to four-fold in white spot syndrome virus (WSSV) infected hemocytes at 48 h. Interestingly, PmCypA protein was only detected extracellularly in shrimp plasma at 24 h post WSSV infection. To find out the function of extracellular PmCypA, the recombinant PmCypA (rPmCypA) was produced and administrated in shrimp primary hemocyte cell culture to observe the antiviral properties. In rPmCypA-administrated hemocyte cell culture, the mRNA transcripts of WSSV intermediate early gene, ie1 and early gene, wsv477 were significantly decreased but not that of late gene, vp28. To explore the antiviral mechanism of PmCypA, the expression of PmCypA in shrimp hemocytes was silenced and the expression of immune-related genes were investigated. Surprisingly, the suppression of PmCypA affected other gene expression, decreasing of penaeidin, PmHHAP and PmCaspase and increasing of C-type lectin. Our results suggested that the PmCypA might plays important role in anti-WSSV via apoptosis pathway. Further studies of PmCypA underlying antiviral mechanism are underway to show its biological function in shrimp immunity., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Importance: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination., Competing Interests: N.S., P.K., N.C., and M.S.-L. were listed as inventors in patents and patent applications involving the mature virus-like particles of flaviviruses. Other authors declare that they do not have conflicts of interest.

Humans infected with dengue virus (DENV) acquire long-term protection against the infecting serotype, whereas cross-protection against other serotypes is short-lived. Long-term protection induced by low levels of type-specific neutralizing antibodies can be assessed using the virus-neutralizing antibody test. However, this test is laborious and time-consuming. In this study, a blockade-of-binding enzyme-linked immunoassay was developed to assess antibody activity by using a set of neutralizing anti-E monoclonal antibodies and blood samples from dengue virus-infected or -immunized macaques. Diluted blood samples were incubated with plate-bound dengue virus particles before the addition of an enzyme-conjugated antibody specific to the epitope of interest. Based on blocking reference curves constructed using autologous purified antibodies, sample blocking activity was determined as the relative concentration of unconjugated antibody that resulted in the same percent signal reduction. In separate DENV-1-, -2-, -3-, and -4-related sets of samples, moderate to strong correlations of the blocking activity with neutralizing antibody titers were found with the four type-specific antibodies 1F4, 3H5, 8A1, and 5H2, respectively. Significant correlations were observed for single samples taken 1 month after infection as well as samples drawn before and at various time points after infection/immunization. Similar testing using a cross-reactive EDE-1 antibody revealed a moderate correlation between the blocking activity and the neutralizing antibody titer only for the DENV-2-related set. The potential usefulness of the blockade-of-binding activity as a correlative marker of neutralizing antibodies against dengue viruses needs to be validated in humans. IMPORTANCE This study describes a blockade-of-binding assay for the determination of antibodies that recognize a selected set of serotype-specific or group-reactive epitopes in the envelope of dengue virus. By employing blood samples collected from dengue virus-infected or -immunized macaques, moderate to strong correlations of the epitope-blocking activities with the virus-neutralizing antibody titers were observed with serotype-specific blocking activities for each of the four dengue serotypes. This simple, rapid, and less laborious method should be useful for the evaluation of antibody responses to dengue virus infection and may serve as, or be a component of, an in vitro correlate of protection against dengue in the future., Competing Interests: The authors declare no conflict of interest.

Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, has become a pandemic and public health crisis. SARS-CoV-2 and the seasonal common cold coronavirus (HCoV-OC43) belong to the beta genus of human coronaviruses (HCoVs). In-cell ELISA assays were performed using HCoV-OC43 and SARS-CoV-2 and evaluated the antiviral activity of herbal plants. Eurycoma longifolia (EL) and Eurycoma harmandiana (EH) roots (antipyretic properties) and their constituent quassinoids, especially chaparrinone and eurycomalactone, showed potent anti-HCoV-OC43 and SARS-CoV-2 activities, and the low IC 50 values of the mentioned constituents were observed in the range of 0.32-0.51 μM. Eurycomanone and 13β,21-dihydroeurycomanone may contribute to the antiviral activity of EL, whereas chaparrinone is the major and active antiviral constituent of EH root. The content of quassinoids, β - carboline, and canthin-6-one alkaloids and the cytotoxicity profile of EL and EH extracts were varied regarding extraction solvents. The boiled water and 50% EtOH extractions of both plants were less toxic than those with 95% EtOH as the extraction solvent. Our research suggests that quassinoids, which come from EL and EH roots and are anti-coronavirus compounds, are potential treatment candidates for COVID-19 and merit further in vivo investigations.

Concerns have been raised about viral contamination, including in crops due to the recent coronavirus disease 2019 pandemic. Limited evidence is available to support the use of sanitizing agents for human coronavirus-contaminated medicinal plants. Thus, we aimed to investigate the persistence of infectious human coronavirus OC43 (HCoV-OC43) as a SARS-CoV-2 surrogate in storage conditions and the capability of neutral electrolyzed water (NEW) to inactivate coronavirus, including in fresh plants such as C. asiatica . The levels of infectious HCoV-OC43 and the triterpenoid content of C. asiatica were quantified using a plaque assay and high-performance liquid chromatography, respectively. The results showed that the persistence of HCoV-OC43 on C. asiatica leaves is identical to that on inert polystyrene. When covered and kept at room temperature with high humidity (>90% RH), HCoV-OC43 can be stable on C. asiatica leaves for at least 24 h. NEW with 197 ppm of available chlorine concentration (ACC) was effective in inactivating both infectious HCoV-OC43 and SARS-CoV-2 in suspension (≥3.68 and ≥4.34 log reduction, respectively), and inactivated dried HCoV-OC43 on the surfaces of C. asiatica leaves (≥2.31 log reduction). Soaking C. asiatica leaves for 5 min in NEW with 205 ppm of ACC or water resulted in significantly higher asiaticoside levels (37.82 ± 0.29 and 35.32 ± 0.74 mg/g dry weight, respectively), compared to the unsoaked group (29.96 ± 0.78 mg/g dry weight). These findings suggest that although coronavirus-contaminated C. asiatica leaves can pose a risk of transmission, NEW could be an option for inactivation., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Although discrimination between primary and secondary dengue infections can be performed using commercially available immunoassays or in-house tests, the evaluation of these methods is important, but is often problematic due to incomplete clinical data. In many cases, patients' sera submitted to the laboratory may not include the date of onset of illness which is necessary to discriminate primary and secondary dengue infections. This study reports improvement of an in-house capture ELISA using IgG avidity to discriminate primary and secondary dengue virus infection. Modified definition criteria were applied to characterize 99 single sera based on their IgM/IgG ratios. Regressive analysis indicated that the avidity test results (avidity index of 60 % as cutoff) for the discrimination showed good agreement (96 %) and a high correlation (r = -0.81) with those of the in-house capture ELISA (IgM/IgG ratio at 1.2 as cutoff). To further evaluate the in-house tests, 318 convalescent sera were compared with a Focus Diagnostics' anti-dengue IgM ELISA. Compared with the Focus Diagnostics system, the sensitivity of an in-house IgM determination was 83 %, whereas using both IgM and IgG capture ELISAs the sensitivity increased to 95 %., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Spätzle is a signaling ligand in innate immune response that signals pathogenic infection via Toll receptor and Toll pathway into the cells for the synthesis of antimicrobial proteins. Herein, three PmSpӓtzle isoforms were identified in Penaeus monodon, namely PmSpz1, 2 and 3. The PmSpz1 was chosen for detailed study. The PmSpz1 gene was expressed in all nine tissues tested including the hemocytes, stomach, hepatopancreas, gill, lymphoid tissue, eyestalk, muscle, intestine and heart. Its expression was up-regulated upon white spot syndrome virus (WSSV) infection. Western blot analysis of hemolymph showed that the PmSpz1 mostly existed as a cleaved active form awaiting to activate the Toll pathway. Injection of a recombinant PmSpz1 rendered the shrimp less susceptible to the WSSV infection. Injection of a recombinant active form of PmSpz1 into a normal shrimp activated the synthesis of crustinPm1, crustinPm7, ALFPm3, penaeidin3 but not penaeidin5 indicating that the expression of all antimicrobial proteins but not penaeidin5 was under the regulation of Toll pathway., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Chikungunya virus (CHIKV) causes an acute clinical illness characterized by sudden high fever, intense joint pain, and skin rash. Recent outbreaks of chikungunya disease in Africa and Asia are a major public health concern; however, there is currently no effective licensed vaccine or specific treatment. This study reported the development of a mouse monoclonal antibody (MAb), CK47, which recognizes domain III within the viral envelope 1 protein and inhibited the viral release process, thereby preventing the production of progeny virus. The MAb had no effect on virus entry and replication processes. Thus, CK47 may be a useful tool for studying the mechanisms underlying CHIKV release and may show potential as a therapeutic agent., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The immune response to dengue virus (DENV) infection generates high levels of antibodies (Abs) against the DENV non-structural protein 1 (NS1), particularly in cases of secondary infection. Therefore, anti-NS1 Abs may play a role in severe dengue infections, possibly by interacting (directly or indirectly) with host factors or regulating virus production. If it does play a role, NS1 may contain epitopes that mimic those epitopes of host molecules. Previous attempts to map immunogenic regions within DENV-NS1 were undertaken using mouse monoclonal Abs (MAbs). The aim of this study was to characterize the epitope regions of nine anti-NS1 human monoclonal Abs (HuMAbs) derived from six patients secondarily infected with DENV-2. These anti-NS1 HuMAbs were cross-reactive with DENV-1, -2, and -3 but not DENV-4. All HuMAbs bound a common epitope region located between amino acids 221 and 266 of NS1. This study is the first report to map a DENV-NS1 epitope region using anti-DENV MAbs derived from patients., (© The American Society of Tropical Medicine and Hygiene.)

The non-structural protein NS2B/NS3 serine-protease complex of the dengue virus (DENV) is required for the maturation of the viral polyprotein. Dissociation of the NS2B cofactor from NS3 diminishes the enzymatic activity of the complex. In this study, we identified a small molecule inhibitor that interferes with the interaction between NS2B and NS3 using structure-based screening and a cell-based viral replication assay. A library containing 661,417 small compounds derived from the Molecular Operating Environment lead-like database was docked to the NS2B/NS3 structural model. Thirty-nine compounds with high scores were tested in a secondary screening using a cell-based viral replication assay. SK-12 was found to inhibit replication of all DENV serotypes (EC50=0.74-4.92 μM). In silico studies predicted that SK-12 pre-occupies the NS2B-binding site of NS3. Steady-state kinetics using a fluorogenic short peptide substrate demonstrated that SK-12 is a noncompetitive inhibitor against the NS2B/NS3 protease. Inhibition to Japanese encephalitis virus by SK-12 was relatively weak (EC50=29.81 μM), and this lower sensitivity was due to difference in amino acid at position 27 of NS3. SK-12 is the promising small-molecule inhibitor that targets the interaction between NS2B and NS3., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Dengue virus (DENV) infection induces a strong B-cell immune response against the viral nonstructural protein 1 (NS1). Anti-NS1 antibodies (Abs) may affect virus production because they coexist with the virus in the patients' blood. The present study examined whether ten mouse monoclonal antibodies (MAbs) raised against NS1 affected production of the DENV-2. Three MAbs, 4C2, 4G11, and 4E5, showed weak neutralizing activity in a focus reduction assay. In addition, two serotype-specific MAbs, 4C2 and 4G11, protected suckling mice from lethal infection with DENV-2. An immunoprecipitation assay with DENV-2 showed that these MAbs, which were specific for the NS1 of DENV-4 and DENV-1, cross-reacted with the DENV-2 pre-membrane (prM) protein, but not with DENV-2 NS1. Interestingly, high concentrations of MAb 4G11 showed antibody-dependent enhancement of DENV-2 infection in human monocyte THP-1 cells. Taken together, these observations suggest that serotype-specific anti-NS1 MAbs are potentially involved in virus production.

Dengue virus (DENV) is a mosquito-borne virus and can be transmitted to humans by mosquito vectors. Although surveillance of dengue virus-infected mosquitoes is the most effective way of controlling DENV infections, detection of DENVs in mosquitoes is limited by the low sensitivity of available detection methods. We here report a method for capturing DENV type 3 (DENV-3) from mosquito cells using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydrate). The beads were incubated with cell culture medium of DENV-3-infected mosquito cells, then separated from the supernatant by applying a magnetic field and washed. Adsorption of DENV-3 on the beads was confirmed by reverse transcription-polymerase chain reaction, which detected the presence of DENV-3 genomic RNA on the beads, and Western blotting, which determined the major DENV-3 envelope protein on the beads. Therefore, this capture method may enable an improvement in DENV-3 detection.

Dengue virus (DENV) nonstructural protein 1 (NS1) is a major target of humoral immunity in patients and is believed to be involved in DENV pathogenesis. In addition, NS1 is a diagnostic target as it is secreted, and circulates, in patients' plasma at an early stage of viral infection. In this study, we aimed to identify common epitope regions for all serotypes by preparation of mouse monoclonal antibodies (MAbs) against NS1. A total of 10 out of the 20 hybridoma clones which were specific to DENV produced MAbs that recognized NS1. These MAbs mapped to three regions of DENV-2 NS1, namely amino acids 1‒40 (epitope region 1), 141‒168 (epitope region 2), and 267‒312 (epitope region 3). Epitope region 2 was recognized by both complex-specific (2H11 and 3C4) and subcomplex-specific MAbs (4E5 and 5G12), whereas epitope regions 1 and 3 were recognized by subcomplex-specific MAbs (5E2, 1A5, and 3F10) only. These epitope regions were found to be highly conserved among all four serotypes of DENV by sequence analysis and database comparison. The MAbs against these epitope regions, especially 2H11 and 3C4, could therefore be valuable diagnostic tools.

The pathogenic mechanism of the severe form of dengue is complicated. Recent reports indicate that apoptotic death of various tissues or organs may be associated with vascular leakage, and ultimately leads to the death of DENV-infected patients. In the present study, we provide additional evidence supporting the detrimental role of apoptosis in DENV infection. A comparison of the rate of apoptosis in PBMCs isolated from patients suffering DF, a mild form of the disease, and the rate in patients with DHF, a life-threatening disease, revealed that PBMCs from DHF patients underwent apoptosis at a significantly higher rate than those suffering from DF alone. This suggests that the severity of natural DENV infection correlates with PBMC apoptosis. In addition, this cell death was induced not only by DENV itself, but also by the apoptotic activities of pro-inflammatory cytokines, such as TNF-alpha, and IL-1beta, that were upregulated in DHF patients. The death of these mononuclear cells that function in an innate immune system may explain the higher viral load in DHF patients than in DF patients. Interestingly, a gene expression profile pattern elucidated that apoptosis occurring during natural DENV infection involved mainly the extrinsic apoptosis pathway, which is mediated via both caspase-dependent and caspase-independent mechanisms. In conclusion, our data highlight the adverse effect of apoptosis induced by DENV and by pro-inflammatory cytokines during natural DENV infection.

Background: Dengue virus infection causes an array of symptoms ranging from dengue fever (DF) to dengue hemorrhagic fever (DHF). The pathophysiological processes behind these 2 clinical manifestations are unclear., Method: In the present study, genomewide transcriptomes of peripheral blood mononuclear cells (PBMCs) collected from children with acute-phase DF (i.e., DF PBMCs) or acute-phase DHF (i.e., DHF PBMCs) were compared using microarray analysis. Results of genome screening were validated at the genomic and proteomics levels., Results: DHF had stronger influences on the gene expression profile than did DF. Of the affected genes, metabolic gene expression was influenced the most. For the immune response category, 17 genes were more strongly up-regulated in DF PBMCs than in DHF PBMCs. Eight of the these 17 genes were categorized as belonging to the interferon (IFN) system. The up-regulation of IFN-related genes was accompanied by strong expression of CD59, a complement inhibitor. DHF PBMCs expressed genes involved in T and B cell activation, cytokine production, complement activation, and T cell apoptosis more strongly than did DF PBMCs., Conclusion: We hypothesize that, during DF, genes in the IFN system and complement inhibitor play a role in lowering virus production and reducing tissue damage. In patients with DHF, the dysfunction of immune cells, complement, and cytokines increases viral load and tissue damage.

Background: VR3848 is a novel cycloheptapeptide, isolated from a Euphorbiaceae plant, which can suppress proliferation of various tumor cells at nanomolar concentration. Due to its novelty and potency, the molecular process of tumor cell growth inhibition was investigated intensively., Materials and Methods: MCF-7 cells, a caspase-3 deficient cancer cell line, were treated with VR3848. The genetic response was monitored using cDNA array analysis., Results: Expression alterations of caspase, bcl-2 family members, death receptor, death adaptor, death ligands, stress response, cell cycle machinery, mitogen-activated protein kinases (MAPKs) and proto-oncogene were found which can be linked into three apoptotic pathways. The first was the death receptor-mediated pathway, which was confirmed by functional inhibition of caspase-8 and -10. The second pathway was via ER-stress apoptosis demonstrated by up-regulation of ER-stress genes and the release of caspase-12 into the cytoplasm. The third pathway involved mitochondrial membrane leakage which was elucidated by down-regulation of anti-apoptotic bcl-2 and an increased level of cytosolic apoptosis-inducing factor (AIF). Cell cycle arrest was observed which may have been a direct effect of VR3848 or a consequence of DNA strand breaks which in turn stimulated cell cycle arrest., Conclusion: VR3848 inhibited MCF-7 cancer cell growth through an activation of three related apoptotic pathways.