Sa-nga Pattanakitsakul

Vascular Leakage in Severe Dengue Virus Infections: A Potential Role for the Nonstructural Viral Protein NS1 and Complement

BACKGROUND Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. METHODS The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed. RESULTS Soluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS. CONCLUSIONS Complement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS.

Renal tubular function in β-thalassemia

Abstract. Studies of the renal involvement in thalassemic syndromes have been varied and few. This study was designed to define the renal abnormalities associated with β-thalassemia and to correlate the renal findings with clinical parameters. One hundred and four β-thalassemic children with various disease severity were studied. The patients were divided into three groups: 48 with severe anemia [hematocrit (Hct) <25%], 31 on a hypertransfusion program and desferrioxamine treatment, and 25 with moderate anemia (Hct >25%). The results were compared with 15 normal children. Significantly higher levels of proteinuria and low molecular weight proteinuria were found in all patients compared with normal children. Aminoaciduria was detected in one-third of patients. Thalassemic patients had significantly lower morning urine osmolarity, higher urine N-acetyl-β-D-glucoseminidase and malondialdehyde (MDA, an indicator of lipid peroxidation). Patients with severe anemia had significantly higher low-molecular weight proteinuria and MDA, and lower urine osmolarity than those with moderate anemia. Our data confirmed the high frequency of renal abnormalities in β-thalassemia patients and indicated some degree of proximal tubular dysfunction. Severity of the abnormalities correlated with the degree of anemia and were least severe in patients on hypertransfusion and desferrioxamine therapy. This suggested that the damage might be caused by anemia and increased oxidation induced by excess iron deposits.

Alterations in actin cytoskeletal assembly and junctional protein complexes in human endothelial cells induced by dengue virus infection and mimicry of leukocyte transendothelial migration.

Vascular leakage is a hallmark of severe dengue infection. Although extensive studies have been conducted during the past several decades, the molecular mechanisms underlying vascular leakage in dengue shock syndrome (DSS) remain unclear. We thus performed a proteomics study to characterize responses in human endothelial cells (EA.hy926) after DEN-2 virus infection (MOI=10). Comparative 2-D PAGE analysis revealed significantly altered abundance levels of 15 proteins, which were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS). These altered proteins were involved in several biological processes, for example, mRNA stability/processing, transcription and translation regulation, molecular chaperoning, oxidative stress response/regulation, cytoskeletal assembly, protein degradation, and cellular metabolisms. We also performed functional analyses of alterations in actin cytoskeletal assembly and endothelial integrity focusing on adherens junction (VE-cadherin), tight junction (ZO-1) and adhesive molecule (PECAM-1) after 24-h of DEN-2 infection and simulation of transendothelial migration by PECAM-1 cross-linking. Decreased expression and disorganization of the actin-cytoskeleton were observed in the infected cells, whereas the increase in actin stress fibers was found in adjacent noninfected cells. Additionally, a decrease in adhesive protein PECAM-1 was observed. Furthermore, DEN-2 infection caused decreased expression and redistribution of both VE-cadherin and ZO-1, whose changes were enhanced by PECAM-1 engagement. These alterations may potentially be a molecular basis explaining increased endothelial permeability or vascular leakage in DSS.

The Ubiquitin−Proteasome Pathway Is Important for Dengue Virus Infection in Primary Human Endothelial Cells

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the most severe forms of dengue virus infection with hemorrhage and plasma leakage. However, pathogenic mechanisms of DHF and DSS remain poorly understood. We therefore investigated host responses as determined by changes in the cellular proteome of primary human endothelial cells upon infection with dengue virus serotype 2 (DEN-2) at a multiplicity of infection (MOI) of 10 for 24 h. Two-dimensional PAGE and quantitative intensity analysis revealed 38 significantly altered protein spots (16 upregulated and 22 downregulated) in DEN-2-infected cells compared to mock controls. These altered proteins were successfully identified by mass spectrometry, including those involved in oxidative stress response, transcription and translation, cytoskeleton assembly, protein degradation, cell growth regulation, apoptosis, cellular metabolism, and antiviral response. The proteomic data were validated by Western blot analyses [upregulated ubiquitin-activating enzyme E1 (UBE1) and downregulated annexin A2] and an immunofluorescence study (upregulated MxA). Interestingly, we found that MxA was colocalized with DEN-2 viral capsid protein, strengthening its role as an antiviral protein. Moreover, we also identified upregulation of a proteasome subunit. Our functional study revealed the significant role of ubiquitination in dengue infection and UBE1 inhibition by its specific inhibitor (UBEI-41) caused a significant reduction in the level of viral protein synthesis and its infectivity. Our findings suggest that various biological processes were triggered in response to dengue infection, particularly antiviral IFN and ubiquitin-proteasome pathways.

Vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein 1 and is important for viral replication and release

Our previous study using expression proteomics demonstrated that many proteins, particularly five forms of heterogeneous nuclear ribonucleoproteins (hnRNPs), were up-regulated in human endothelial cells upon dengue virus infection. To address functional significance of these proteins in response to dengue virus infection, we performed a functional proteomics study to identify hnRNPs-interacting proteins in the infected EA.hy926 cells. Immunoprecipitation followed by 2-D PAGE and mass spectrometric analyses revealed 18 and 13 interacting partners of hnRNP C1/C2 and hnRNP K, respectively. Interestingly, vimentin was a common partner for both hnRNP C1/C2 and K. The interaction between vimentin and these hnRNPs was confirmed by reciprocal immunoprecipitation followed by Western blot analysis and also by double immunofluorescence staining. Disruption of vimentin intermediate filament by acrylamide not only dissociated these complexes but also reduced nuclear hnRNPs expression, whereas cytosolic hnRNPs expression was unchanged. We also demonstrated that vimentin was strongly associated with dengue non-structural protein 1 (NS1). Disruption of vimentin intermediate filament not only dissociated this complex but also reduced dengue NS1 expression, as well as viral replication and release. Our data report for the first time that vimentin interacts with hnRNPs and dengue NS1, and plays a crucial role in replication and release of dengue virus.

Mosquito cells accommodate balanced, persistent co-infections with a densovirus and Dengue virus

To study persistent viral co-infections in arthropods, we first produced stable, persistently infected C6/36 mosquito cell cultures by serial passage of exponentially growing whole cells infected with either a densovirus (AalDNV) or Dengue virus (DEN-2). We then obtained stable, persistent co-infections by reciprocal super-challenge and similar passaging. Persistently infected cultures did not differ from naïve-cell cultures in growth rate and cell morphology. Nor did they differ in high production of both viruses with high infection rates for naïve C6/36 cells. Immunocytochemistry revealed that 99-100% of the cells were coinfected but that super-infection order had some effect on antigen distribution for the two viruses. Our results combined with existing field information and previously published experimental work suggest that the capacity to support stable, viral co-infections may be a general phenomenon for arthropod cells, and that they may be achieved easily and rapidly by serial passaging of whole cultured cells. Such persistent infections would facilitate studies on interactions between co-infecting viruses.

Comparison of penaeid shrimp and insect parvoviruses suggests that viral transfers may occur between two distantly related arthropod groups

The DNA and putative amino acid sequences of representative insect and shrimp parvoviruses (subfamily Densovirinae) were analyzed using computer programs. Shrimp viruses included hepatopancreatic parvovirus (HPV) of Penaeus monodon (HPVmon) and P. chinensis (HPVchin), spawner-isolated mortality virus from P. monodon (SMVmon) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) from P. vannamei. Insect viruses included Aedes aegypti densovirus (AaeDNV), Aedes albopictus densovirus (AalDNV), Junonia coenia densovirus (JcDNV), Galleria mellonella densovirus (GmDNV), Bombyx mori densovirus 5 (BmDNV), Diatraea saccharalis densovirus (DsDNV) and Periplaneta fuliginosa densovirus (PfDNV). Virion size for all these viruses ranged between 18 and 30 nm diameter and ssDNA genome length was between 4 and 6 kb. Using BLAST or Clustal W with the sequence fragments available, no significant DNA homology was found except for 77% DNA identity between HPVmon and HPVchin. However, phylogenetic trees constructed by comparing DNA genome sequences for putative viral polypeptides, capsid proteins and nonstructural proteins placed the parvoviruses into two Clades: Clade 1 with SMVmon, PfDNV, DsDNV, GmDNV, JcDNV, and BmDNV; and Clade 2 with HPVmon, HPVchin, IHHNV, AalDNV and AaeDNV. The four shrimp parvoviruses fell into two different clades that grouped with different insect parvoviruses.

Persistent, triple-virus co-infections in mosquito cells

BackgroundIt is known that insects and crustaceans can carry simultaneous, active infections of two or more viruses without showing signs of disease, but it was not clear whether co-infecting viruses occupied the same cells or different cells in common target tissues. Our previous work showed that successive challenge of mosquito cell cultures followed by serial, split-passage resulted in stabilized cultures with 100% of the cells co-infected with Dengue virus (DEN) and an insect parvovirus (densovirus) (DNV). By addition of Japanese encephalitis virus (JE), we tested our hypothesis that stable, persistent, triple-virus co-infections could be obtained by the same process.ResultsUsing immunocytochemistry by confocal microscopy, we found that JE super-challenge of cells dually infected with DEN and DNV resulted in stable cultures without signs of cytopathology, and with 99% of the cells producing antigens of the 3 viruses. Location of antigens for all 3 viruses in the triple co-infections was dominant in the cell nuclei. Except for DNV, this differed from the distribution in cells persistently infected with the individual viruses or co-infected with DNV and DEN. The dependence of viral antigen distribution on single infection or co-infection status suggested that host cells underwent an adaptive process to accommodate 2 or more viruses.ConclusionsIndividual mosquito cells can accommodate at least 3 viruses simultaneously in an adaptive manner. The phenomenon provides an opportunity for genetic exchange between diverse viruses and it may have important medical and veterinary implications for arboviruses.

Association of Alix with late endosomal lysobisphosphatidic acid is important for dengue virus infection in human endothelial cells.

The most severe form of dengue virus (DENV) infection is dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is accompanied by increased vascular permeability indicating that endothelial cells are the targets of DENV infection. However, molecular mechanisms underlying DENV replication in endothelial cells remained poorly understood. We therefore examined changes in subcellular proteomes of different cellular compartments (including cytosolic, membrane/organelle, nucleus, and cytoskeleton) of human endothelial (EA.hy926) cells upon DENV2 infection using a 2-DE-based proteomics approach followed by Q-TOF MS and MS/MS. A total of 35 altered proteins were identified in these subcellular locales, including an increase in the level of Alix (apoptosis-linked gene-2-interacting protein X) in the cytosolic fraction of DENV2-infected cells compared to mock control cells. Double immunofluorescence staining revealed colocalization of Alix with late endosomal lysobisphosphatidic acid (LBPA). This complex has been proposed to be involved in the export of DENV proteins from late endosomes to the cytoplasm. Subsequent functional study revealed that pretreatment with an anti-LBPA antibody prior to DENV challenge significantly reduced the level of viral envelope protein synthesis and DENV replication. Our data indicate that Alix plays a pivotal role in the early phase of DENV replication, particularly when it arrives at the late endosome stage. Blocking this step may lead to a novel therapeutic approach to reducing the level of DENV replication in vivo.

Heterogeneity in filterability of erythrocytes from malaria (Plasmodium berghei)-infected blood

Erythrocytes fromPlasmodium berghei-infected blood show a decrease in deformability with increasing parasitaemia, as measured by filterability through polycarbonate sieves. A major fraction of cells carrying mature parasites and a smaller fraction carrying ring-stage parasites account for the obstruction of filtration, while the remaining infected cells do not contribute to the decrease in filterability. The relation of filterability to metabolic status in infected cells is discussed.