Activation of dengue virus-specific T cells modulates vascular endothelial growth factor receptor 2 expression

Background: The pathogenic mechanisms underlying the increased vascular permeability in dengue hemorrhagic fever (DHF) are not well understood. Enhanced cellular immune activation, especially activation of serotype-cross reactive T cells, has been implicated in plasma leakage in DHF. Changes in several biological markers and mediators including cytokines, chemokines, angiogenic factors and their receptors have been shown to correlate with disease severity. A decline in plasma levels of a soluble form of vascular endothelial growth factor receptor 2 (VEGFR2), a receptor of vascular endothelial growth factor (VEGF), has been associated with plasma leakage in dengue patients. Objective: We aimed to investigate the effect of dengue virus (DV)-specific CD8 + T cells on the expression of VEGFR2 on endothelial cells. Method: An in vitro model was developed in which dengue virus-specific CD8 + T cells generated from peripheral blood mononuclear cells (PBMCs) of DHF patients were co-cultured with antigen-presenting cells, human umbilical vein endothelial cells (HUVECs) and activated with DV non-structural protein 3 (NS3) peptides. The expression of VEGFR2 by endothelial cells was measured. Results: DV-specific CD8 + T cells were serotype cross-reactive. Activation of DV-specific CD8 + T cells resulted in down-regulation of soluble VEGFR2 production and an up-regulation of cell-associated VEGFR2. Conclusions: Our findings indicate that activation of DV-specific T cell is associated with modulation of VEGFR2 expression that may contribute to increased VEGF responsiveness and vascular permeabilit

Inhibition of N ‐myristoyltransferase1 affects dengue virus replication

International audienceDengue virus (DENV) causes dengue fever, a self‐limiting disease that could be fatal due to serious complications. No specific treatment is currently available and the preventative vaccine is only partially protective. To develop a potential drug target for dengue fever, we need to understand its biology and pathogenesis thoroughly. N‐myristoyltransferase (NMT) is an N‐terminal protein lipidation enzyme that catalyzes the covalent cotranslational attachment of fatty acids to the amino‐terminal glycine residue of a number of proteins, leading to the modulation of various signaling molecules. In this study, we investigated the interaction of dengue viral proteins with host NMT and its subsequent effect on DENV. Our bioinformatics, molecular docking, and far‐western blotting analyses demonstrated the interaction of viral envelope protein (E) with NMT. The gene expression of NMT was strongly elevated in a dependent manner during the viral replication phase in dendritic cells. Moreover, NMT gene silencing significantly inhibited DENV replication in dendritic cells. Further studies investigating the target cell types of other host factors are suggested

Diagnostic Performance of Dengue NS1 and Antibodies by Serum Concentration Technique

Dengue infection has been a public health problem worldwide, especially in tropical areas. A lack of sensitive diagnostic methods in the early phase of the illness is one of the challenging problems in clinical practices. We, herein, analyzed 86 sera of acute febrile patients, from both dengue and non-dengue febrile illness, to study the diagnostic performance of dengue diagnostics. When compared with detection by Polymerase Chain Reaction (PCR), dengue NS1 detection by enzyme-linked immunosorbent assay (ELISA) had the highest sensitivity of 82.4% (with 94.3% specificity), while NS1 by rapid diagnostic test (RDT) had 76.5% sensitivity. IgM detection by ELISA and RDT showed only 27.5% and 17.9% sensitivity, respectively. The combination of NS1 and IgM in RDT yielded a sensitivity of 78.4%, with 97.1% specificity. One of the essential steps in making a diagnosis from patient samples is the preparation process. At present, a variety of techniques have been used to increase the number of analytes in clinical samples. In this study, we focused on the sample concentration method. The sera were concentrated three times with the ultrafiltration method using a 10 kDa molecular weight cut-off membrane. The results showed an increase in the sensitivity of RDT-NS1 detection at 80.4%, with 100% specificity. When combining NS1 and IgM detection, the concentration method granted RDT an 82.4% sensitivity, with 100% specificity. In conclusion, serum concentration by the ultrafiltration method is a simple and applicable technique. It could increase the diagnostic performance of point-of-care dengue diagnostics

A Use of 56-kDa Recombinant Protein of <i>Orientia tsutsugamushi</i> Karp Serotype in Serodiagnosis of Scrub Typhus by Enzyme-Linked Immunosorbent Assay in Thais

Scrub typhus is a mite-borne disease caused by a Gram-negative obligately intracellular bacillus, Orientia tsutsugamushi. The disease is endemic in the Asia–Australia–Pacific region, including Thailand. Scrub typhus generally manifests as acute undifferentiated febrile fever along with myalgia, rash, and lymphadenopathy. An eschar can be a valuable diagnostic clue, but this skin lesion may be missed in some patients. The disease symptoms resemble those of other febrile illnesses such as leptospirosis, typhoid, murine typhus, malaria, and dengue fever, making a laboratory diagnosis necessary for the definitive diagnosis. In this study, we expressed a recombinant protein derived from 56-kDa type-specific antigen of O. tsutsugamushi Karp serotype and tested its ability to detect and differentiate scrub typhus infection. IgM and IgG antibodies were determined in sera from scrub typhus (n = 92) and other febrile illness patients (murine typhus (n = 25), melioidosis (n = 36), leptospirosis (n = 42), and dengue (n = 35)) from Thailand. Sensitivities of 87.0% and 59.8% with a specified assay cut-off were obtained for IgM and IgG indirect ELISAs, respectively, with a specificity of 100% in both tests. The sensitivity was increased to 95.7% when a combination of IgM and IgG ELISAs results was considered. Our study suggested a potential of the 56-kDa recombinant protein for further development and evaluation for use in scrub typhus serodiagnosis

Detection of SARS-CoV-2 and Variants in Hospital Wastewater in a Developing Country

Wastewater-based epidemiology (WBE) is a beneficial tool for comprehensive health information on communities, especially during the COVID-19 pandemic. In developing countries, including Thailand, the application of WBE is limited. Few SARS-CoV-2 detections and variants have been monitored in wastewater in these countries. This is because of the time-consuming, low recovery of viruses in the concentration techniques and difficulties in finding the proper primers and amplification kits. Therefore, this study aimed to quantify SARS-CoV-2 RNA concentration using a commercial clinical kit. We identified the SARS-CoV-2 variants and estimated the detection costs in the wastewater samples. One hundred and fifty hospital wastewater samples were filtered with commercial ultrafiltration (UF) and then detected for the SARS-CoV-2 concentration using a Sansure Biotech SARS-CoV-2 kit. The recovery of the virus concentration technique in UF was studied using a surrogate (porcine epidemic diarrhea virus). The virus detection in wastewater was quantified by RT-qPCR. In addition, the mutation sites in the partial spike glycoprotein (S) gene of SARS-CoV-2 were verified using short nested RT-PCR. The results showed a high recovery of the commercial UF (80.53%), and 24.6% of hospital wastewater contained SARS-CoV-2. The detection of SARS-CoV-2 in wastewater cost USD 35.43 per sample. The virus variants revealed V70del, H69del, and V144del mutations in the partial S gene of SARS-CoV-2 in B.1.1.7 (SARS-CoV-2 Alpha variant), and T95I and G142D mutations in B.1.617.2 (Delta variant)

Leaky Gut Syndrome Is Associated with Endotoxemia and Serum (1→3)-β-D-Glucan in Severe Dengue Infection

The hallmark of severe dengue infection is the increased vascular permeability and hemodynamic alteration that might be associated with an intestinal permeability defect. However, the mechanisms underlying the gastrointestinal-related symptoms of dengue are not well characterized. A prospective observational study was conducted on patients with dengue who were categorized according to: (i) febrile versus critical phase and (ii) hospitalized patients with versus without the warning signs to evaluate the gut barrier using lactulose-to-mannitol excretion ratio (LEMR). Serum endotoxins, (1→3)-β-D-glucan (BG), and inflammatory parameters were measured. A total of 48 and 38 patients were enrolled in febrile illness and critical phase, respectively, while 22 and 64 patients presented with or without the warning signs, respectively. At enrollment, a positive LEMR test was found in 20 patients (91%) with warning signs, regardless of phase of infection. Likewise, serum endotoxins and BG, the indirect biomarkers for leaky gut, prominently increased in patients who developed severe dengue when compared with the non-severe dengue (endotoxins, 399.1 versus 143.4 pg/mL (p &lt; 0.0001); BG, 123 versus 73.8 pg/mL (p = 0.016)). Modest impaired intestinal permeability occurred in dengue patients, particularly those with warning signs, and were associated with endotoxemia and elevated BG. Thus, leaky gut syndrome might be associated with severity of dengue infection

Leaky Gut Syndrome Is Associated with Endotoxemia and Serum (1→3)-β-D-Glucan in Severe Dengue Infection

The hallmark of severe dengue infection is the increased vascular permeability and hemodynamic alteration that might be associated with an intestinal permeability defect. However, the mechanisms underlying the gastrointestinal-related symptoms of dengue are not well characterized. A prospective observational study was conducted on patients with dengue who were categorized according to: (i) febrile versus critical phase and (ii) hospitalized patients with versus without the warning signs to evaluate the gut barrier using lactulose-to-mannitol excretion ratio (LEMR). Serum endotoxins, (1→3)-β-D-glucan (BG), and inflammatory parameters were measured. A total of 48 and 38 patients were enrolled in febrile illness and critical phase, respectively, while 22 and 64 patients presented with or without the warning signs, respectively. At enrollment, a positive LEMR test was found in 20 patients (91%) with warning signs, regardless of phase of infection. Likewise, serum endotoxins and BG, the indirect biomarkers for leaky gut, prominently increased in patients who developed severe dengue when compared with the non-severe dengue (endotoxins, 399.1 versus 143.4 pg/mL (p p = 0.016)). Modest impaired intestinal permeability occurred in dengue patients, particularly those with warning signs, and were associated with endotoxemia and elevated BG. Thus, leaky gut syndrome might be associated with severity of dengue infection

Detection of SARS-CoV-2 and Variants in Hospital Wastewater in a Developing Country

Wastewater-based epidemiology (WBE) is a beneficial tool for comprehensive health information on communities, especially during the COVID-19 pandemic. In developing countries, including Thailand, the application of WBE is limited. Few SARS-CoV-2 detections and variants have been monitored in wastewater in these countries. This is because of the time-consuming, low recovery of viruses in the concentration techniques and difficulties in finding the proper primers and amplification kits. Therefore, this study aimed to quantify SARS-CoV-2 RNA concentration using a commercial clinical kit. We identified the SARS-CoV-2 variants and estimated the detection costs in the wastewater samples. One hundred and fifty hospital wastewater samples were filtered with commercial ultrafiltration (UF) and then detected for the SARS-CoV-2 concentration using a Sansure Biotech SARS-CoV-2 kit. The recovery of the virus concentration technique in UF was studied using a surrogate (porcine epidemic diarrhea virus). The virus detection in wastewater was quantified by RT-qPCR. In addition, the mutation sites in the partial spike glycoprotein (S) gene of SARS-CoV-2 were verified using short nested RT-PCR. The results showed a high recovery of the commercial UF (80.53%), and 24.6% of hospital wastewater contained SARS-CoV-2. The detection of SARS-CoV-2 in wastewater cost USD 35.43 per sample. The virus variants revealed V70del, H69del, and V144del mutations in the partial S gene of SARS-CoV-2 in B.1.1.7 (SARS-CoV-2 Alpha variant), and T95I and G142D mutations in B.1.617.2 (Delta variant)

Spread of a Novel Indian Ocean Lineage Carrying E1-K211E/E2-V264A of Chikungunya Virus East/Central/South African Genotype across the Indian Subcontinent, Southeast Asia, and Eastern Africa

The Indian Ocean Lineage (IOL) of the chikungunya virus (CHIKV) East/Central/South African (ECSA) genotype, which originated in Kenya, spread to the Indian ocean and the Indian subcontinent, and then expanded through Southeast Asia in the previous decade. It carried an adaptive mutation E1-A226V, which enhances CHIKV replication in Aedes albopictus. However, the IOL CHIKV of the most recent outbreaks during 2016&ndash;2020 in India, Pakistan, Bangladesh, the Maldives, Myanmar, Thailand, and Kenya lacked E1-A226V but carried E1-K211E and E2-V264A. Recent CHIKV genome sequences of the Maldives and Thailand were determined, and their phylogenetic relationships were further investigated together with IOL sequences reported in 2004&ndash;2020 in the database. The results showed that the ancestral IOLs diverged to a sub-lineage E1-K211E/E2-V264A, probably in India around 2008, and caused sporadic outbreaks in India during 2010&ndash;2015 and in Kenya in 2016. The massive expansion of this new sub-lineage occurred after the acquisition of E1-I317V in other neighboring and remote regions in 2014&ndash;2020. Additionally, the phylogenetic tree indicated that independent clades formed according to the geographical regions and introduction timing. The present results using all available partial or full sequences of the recent CHIKVs emphasized the dynamics of the IOL sub-lineages in the Indian subcontinent, Southeast Asia, and Eastern Africa

In Vitro and In Vivo Attenuation of Vesicular Stomatitis Virus (VSV) by Phosphoprotein Deletion.

Vesicular stomatitis virus (VSV) is highly immunogenic and able to stimulate both innate and adaptive immune responses. However, its ability to induce adverse effects has held back the use of VSV as a potential vaccine vector. In this study we developed VSV-ΔP, a safe yet potent replication-defective recombinant VSV in which the phosphoprotein (P) gene was deleted. VSV-ΔP replicated only in supporting cells expressing P (BHK-P cells) and at levels more than 2 logs lower than VSV. In vivo studies indicated that the moderate replication of VSV-ΔP in vitro was associated with the attenuation of this virus in the mouse model, whereas mice intracranially injected with VSV succumbed to neurotoxicity. Furthermore, we constructed VSV and VSV-ΔP expressing a variety of antigens including hemagglutinin-neuraminidase (HN) from Newcastle disease virus (NDV), hemagglutinin (HA) from either a 2009 H1N1 pandemic influenza virus (pdm/09) or the avian H7N9. VSV and VSV-ΔP incorporated the foreign antigens on their surface resulting in induction of robust neutralizing antibody, serum IgG, and hemagglutination inhibition (HAI) titers against their corresponding viruses. These results indicated that VSV with P gene deletion was attenuated in vitro and in vivo, and possibly expressed the foreign antigen on its surface. Therefore, the P gene-deletion strategy may offer a potentially useful and safer approach for attenuating negative-sense RNA viruses which use phosphoprotein as a cofactor for viral replication