Background: The mechanisms responsible for thrombocytopenia associated with dengue fever (DF) and dengue hemorrhage fever (DHF) remain unclear. Objective: In this study, we investigated the pathogenic effects of dengue virus (DEN-V) non-structural protein 1 (NS1) on the elicitation of platelet cross-reactive antibodies. Results: The results showed that anti-DENV NS 1 immunoglobulins (Igs) derived from both patients with DF/DHF and recombinant NS1-immunized rabbits could opsonize normal human platelets and enhance platelet-macrophage engagements in vitro. In addition, treatments with anti-NS1 Igs abnormally activated human platelets and induced thrombocytopenia in mice. These prothrombotic characteristics of anti-NS1 Ig might increase the disease burden of coagulant-aberrant DHF patients. To test this hypothesis, we injected anti-NS1 Igs into C57BL/6J mice that were preconditioned into a hypercoagulable state by warfarin treatments. When given before but not after platelet-lysate pre-adsorption, the anti-NS1 Igs injection treatments significantly increased mortality, fibrin deposition in lung, and plasma D-dimer levels, but significantly decreased anticoagulant proteins C, protein S and antithrombin III. Conclusions: These results suggest that the platelet-bound antibody fractions of anti-NS1 Ig are prothrombotic, which might exacerbate the severity of disease in hosts with an imbalanced coagulant system.

During the 2003 outbreak of severe acute respiratory syndrome (SARS) in Taiwan, >150,000 persons were quarantined, 24 of whom were later found to have laboratory-confirmed SARS-coronavirus (SARS-CoV) infection. Since no evidence exists that SARS-CoV is infective before the onset of symptoms and the quarantined persons were exposed but not symptomatic, we thought the quarantine's effectiveness should be investigated. Using the Taiwan quarantine data, we found that the onset-to-diagnosis time of previously quarantined confirmed case-patients was significantly shortened compared to that for those who had not been quarantined. Thus, quarantine for SARS in Taiwan screened potentially infective persons for swift diagnosis and hospitalization after onset, thereby indirectly reducing infections. Full-scale quarantine measures implemented on April 28 led to a significant improvement in onset-to-diagnosis time of all SARS patients, regardless of previous quarantine status. We discuss the temporal effects of quarantine measures and other interventions on detection and isolation as well as the potential usefulness of quarantine in faster identification of persons with SARS and in improving isolation measures.

To date, all human embryonic stem cells (hESCs) available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Activation of STAT3 by leukemia inhibitory factor is required to maintain "stemness" in mouse embryonic stem cells, but not in hESCs, suggesting the existence of alternate signaling pathways for self-renewal and pluripotency in human cells. Here we show that activin A is secreted by mouse embryonic feeder layers (mEFs) and that culture medium enriched with activin A is capable of maintaining hESCs in the undifferentiated state for > 20 passages without the need for feeder layers, conditioned medium from mEFs, or STAT3 activation. hESCs retained both normal karyotype and markers of undifferentiated cells, including Oct-4, nanog, and TRA-1-60 and remained pluripotent, as shown by the in vivo formation of teratomas.

The metabolic actions of insulin are transduced through the phosphatidylinositol 3-kinase pathway. A critical component of this pathway is 3-phosphoinositide-dependent kinase-1 (PDK-1), a PH domain-containing enzyme that catalyzes the activating phosphorylation for many AGC kinases, including Akt and protein kinase C isozymes. We used a directed proteomics-based approach to identify the adaptor protein Grb14, which binds the insulin receptor through an SH2 domain, as a novel PDK-1 binding partner. Interaction of these two proteins is constitutive and mediated by a PDK-1 binding motif on Grb14. Disruption of this motif by point mutation or deletion of the Grb14 SH2 domain prevents the insulin-triggered membrane translocation of PDK-1. The interaction of PDK-1 with Grb14 facilitates Akt function: disruption of the interaction by overexpression of a construct of Grb14 mutated in the PDK-1 binding motif significantly decreases insulin-dependent activation of Akt. Thus, Grb14 serves as an adaptor protein to recruit PDK-1 to activated insulin receptor, thus promoting Akt phosphorylation and transduction of the insulin signal.

Activation and expansion of dengue virus‐specific T cells and abnormal liver functions in dengue patients have been documented. However, it remains to be determined whether T cells are involved in the pathogenic mechanism of dengue virus infection. In this study, immunocompetent C57BL/6 mice were employed to study dengue virus‐induced T cell activation. Mice were inoculated with 10 8 PFU dengue virus serotype 2 strain 16681 by the intravenous route. Dengue viral core RNA was detected by RT‐PCR in mouse serum, liver, spleen, and brain at different time points after infection. Splenic T cells were activated as evidenced by their expression of CD69 and O ‐glycosylated CD43 at as early as day 3 after infection. Splenic T cell expression of O ‐glycosylated CD43 and IFN‐γ production coordinately peaked at day 5. Coincided with the peak of splenic T cell activation was hepatic lymphocyte infiltration and elevation of liver enzymes. Flow cytometric analysis revealed the infiltrating CD8 + T cell to CD4 + T cell ratio was 5/3. After a second inoculation of dengue virus, hepatic T cell infiltration and liver enzyme levels increased sharply. The infiltrating hepatic CD8 + T cell to CD4 + T cell ratio increased to 5.8/1. A strong correlation was found between T cell activation and hepatic cellular infiltration in immunocompetent mice infected with dengue virus. The kinetics of liver enzyme elevation also correlated with that of T cell activation. These data suggest a relationship between T cell infiltration and elevation of liver enzymes. J. Med. Virol. 73:419–431, 2004. © 2004 Wiley‐Liss, Inc.

GEF-H1 is a guanine nucleotide exchange factor for Rho whose activity is regulated through a cycle of microtubule binding and release. Here we identify a region in the carboxyl terminus of GEF-H1 that is important for suppression of its guanine nucleotide exchange activity by microtubules. This portion of the protein includes a coiled-coil motif, a proline-rich motif that may interact with Src homology 3 domain-containing proteins, and a potential binding site for 14-3-3 proteins. We identify GEF-H1 as a binding target and substrate for p21-activated kinase 1 (PAK1), an effector of Rac and Cdc42 GTPases, using an affinity-based screen and localize a PAK1 phosphorylation site to the inhibitory carboxylterminal region of GEF-H1. We show that phosphorylation of GEF-H1 at Ser885 by PAK1 induces 14-3-3 binding to the exchange factor and relocation of 14-3-3 to microtubules. Phosphorylation of GEF-H1 by PAK may be involved in regulation of GEF-H1 activity and may serve to coordinate Rho-, Rac-, and Cdc42-mediated signaling pathways.

ABSTRACT Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.

A variety of intracellular signaling pathways are linked to cell surface receptor signaling through their recruitment by Src homology 2 (SH2)/SH3-containing adapter molecules. p21-activated kinase 1 (PAK1) is an effector of Rac/Cdc42 GTPases that has been implicated in the regulation of cytoskeletal dynamics, proliferation, and cell survival signaling. In this study, we describe the specific interaction of PAK1 with the Grb2 adapter protein both in vitro and in vivo. We identify the site of this interaction as the second proline-rich SH3 binding domain of PAK1. Stimulation of the epidermal growth factor receptor (EGFR) in HaCaT cells enhances the level of EGFR-associated PAK1 and Grb2, although the PAK1-Grb2 association is itself independent of this stimulation. A cell-permeant TAT-tagged peptide encompassing the second proline-rich SH3 binding domain of PAK1 simultaneously blocked Grb2 and activated EGFR association with PAK1, in vitro and in vivo, indicating that Grb2 mediates the interaction of PAK1 with the activated EGFR. Blockade of this interaction decreased the epidermal growth factor-induced extension of membrane lamellae. Thus Grb2 may serve as an important mechanism for linking downstream PAK signaling to various upstream pathways.

ABSTRACT Using reverse transcription-PCR and clonal sequencing of the dengue virus envelope gene derived from the plasma samples of six patients, we reported for the first time that dengue virus circulates as a population of closely related genomes. The extent of sequence diversity varied among patients, with the mean pairwise proportions of difference ranging from 0.21 to 1.67%. Genome-defective viruses were found in 5.8% of the total number of clones analyzed. Our findings on the quasispecies nature of dengue virus and the defective virus in vivo have implications with regard to the pathogenesis of dengue virus.

Objective. In 1998, an enterovirus 71 (EV71) epidemic in Taiwan was associated with hand, foot, and mouth disease (HFMD)/herpangina and involved 78 fatal cases. We measured EV71 seroprevalence rates before and after the epidemic and investigated risk factors associated with EV71 infection and illness.Methods. Neutralizing antibodies to EV71 were assayed for 539 people before the epidemic and 4619 people of similar ages after the epidemic. Questionnaires, which were completed during household interviews after the epidemic, solicited demographic variables, exposure history, and clinical manifestations.Results. A total of 129 106 cases of HFMD were reported during the epidemic. Age-specific pre-epidemic EV71 seroprevalence rates were inversely related to age-specific periepidemic mortality rates (r=-0.82) or severe case rates (r=-0.93). Higher postepidemic EV71 seropositive rates among children who were younger than 3 years positively correlated with higher mortality rates in different areas (r=0.88). After the epidemic, 51 (56%) of 91 younger siblings of elder siblings who were EV71-seropositive were EV71-seropositive; otherwise, 2.2% (4 of 186) of younger siblings were EV71-seropositive (matched odds ratio [OR]: 10; 95% confidence interval [CI]: 3.4-29). Stepwise multiple logistic regression revealed other factors associated with EV71 infection to be older age (adjusted OR: 2.5; 95% CI: 1.9-3.4), attendance at kindergartens/child care centers (adjusted OR: 1.8; 95% CI: 1.3-2.5), contact with HFMD/herpangina (adjusted OR: 1.6; 95% CI: 1.2-2.1), greater number of children in a family (adjusted OR: 1.4; 95% CI: 1.1-1.7), and rural residence (adjusted OR: 1.4; 95% CI: 1.2-1.6). Twenty-nine percent of preschool children who were infected with EV71 developed HFMD/herpangina. Younger age and contact with HFMD/herpangina were significant factors for the development of EV71-related HFMD/herpangina in these children.Conclusions. An increased incidence of EV71 infection in young children occurred more often in geographic areas with increased mortality rates. Intrafamilial and kindergarten transmissions among preschool children were major modes of disease transmission during the widespread EV71 epidemic in Taiwan in 1998.

In dengue virus (DEN) particles, the core protein is a structural protein of the nucleocapsid. The core protein is known to be present in the nucleus of DEN-infected cells but there have been conflicting reports as to whether it is also present in the nucleolus. To clarify this, the intracellular location of the core protein was examined using a monoclonal antibody, 15B11, which was produced in this study. Immunofluorescence staining with this antibody demonstrated that the core protein first appeared in the cytoplasm and then in the nuclei and nucleoli of infected cells. Nuclear localization of the core protein was determined to be independent of other DEN proteins, since recombinant core proteins still entered the nuclei and nucleoli of cells transfected with only the core protein gene. Three putative nuclear localization signal motifs have been predicted to be present on the core protein. Deletion of the first one (KKAR), located at aa 6–9, and mutation of the second one (KKSK), located at aa 73–76, did not eliminate the nuclear localization property of the core protein. The third motif with a bipartite structure, RKeigrmlnilnRRRR, located at aa 85–100, was determined to be responsible for the nuclear localization of the core protein, since the core protein without this motif was located exclusively in the cytoplasm of DEN-infected cells and that this motif mediated nuclear localization of a normally cytoplasmic protein.

ABSTRACT Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60°C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

Background: Influenza surveillance in recent decades in Taiwan showed that the major virus isolates were A/Hong Kong/68-like (H3N2), A/England/42/72-like (H3N2), A/Taiwan/1/86 (H1N1), A/Texas/36/91-like (H1N1), A/Taiwan/112/96 (H1N1), and A/Taiwan/118/96 (H1N1) based on passive virologic surveillance before 1999. Materials and methods: To prevent the potential public health threat of a newly emerged influenza A strain like H5N1 in Hong Kong, seven surveillance systems have been established in Taiwan since 1999, including severe case reporting, sentinel physician, contract-laboratory, poultry market, domestic avian, wild bird and pig surveillance. Scientific exchanges and possible public health actions are discussed in regular meetings during the winter season. Results: The isolation rates of influenza A virus were higher among ducks than chickens in both domestic avian and market surveillance systems. Ten subtypes of influenza A virus can be found in migrating wild birds, which may serve as a reservoir for the genetic and antigenic changes of the virus. The H6 subtype was isolated from both wild birds and market surveillance systems. Pig surveillance found that H3 occurred more in the winter, whereas H1 appeared predominantly in the summer. Human virologic surveillance also demonstrated summer and winter peaks. Market surveillance in Taipei showed H6, H4, and H3 subtypes isolated from ducks only. Sentinel physician surveillance experience has taught us the importance of involving local clinics, particularly in farm areas. The traditional passive physician reporting surveillance provided limited information and it was replaced by severe case reporting. Most elderly physicians in Taiwan did not like to report influenza even after implementation of direct electronic reporting through a worldwide web system. More modern technology will be applied to increase the timeliness and representativeness of influenza surveillance in preparing for future pandemics. Conclusion: Integration of animal and human influenza surveillance systems in the high population density in Taiwan serves as a model in the efforts of global influenza surveillance to detect newly emerged influenza virus strains via several channels, particularly useful in detecting severity of cases and inter-species transmission.

Activation of the mammalian mitogen-activated protein kinase known as BMK1 is required for growth factor-induced cell proliferation. To understand the mechanism by which BMK1 mediates this cellular response, this kinase was used as bait in a yeast two-hybrid-based library screening. Here, we report the identification of serum and glucocorticoid-inducible kinase (SGK) as a cellular protein that physically interacts with BMK1. During growth factor-induced cell stimulation, BMK1 activates SGK by phosphorylation at serine 78. This BMK1-mediated phosphorylation event is necessary for the activation of SGK and, more importantly, for cell proliferation induced by growth factors.

Publisher Summary This chapter explains the purification and in vitro activities of p21-activated kinases (PAKs). The chapter describes various methods for generating recombinant, constitutively active PAKs for substrate phosphorylation studies and wild-type PAKs for kinase activation studies. The chapter discusses the regulation of PAK activity using dominant active mutants or in vitro GTP-labeled forms of Rac and Cdc42, which bind to the p21-binding (PBD) domain. The chapter also discusses the stimulation of PAK kinase activity with lipid compounds that leads to PAK autophosphorylation and substrate phosphorylation with similar kinetics as GTPase stimulation. Identification of PAKs and detection of the PAK activation state has been shown to be achieved in cells by in-gel kinase assays with specific substrates by 32p labeling and tryptic phospho peptide mapping or matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) analysis. The chapter concludes with an analysis of PAK activation and autophosphorylation by MALDI-TOF MS.

In this study, we show that phosphorylated 3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates p21-activated kinase 1 (PAK1) in the presence of sphingosine. We identify threonine 423, a conserved threonine in the activation loop of kinase subdomain VIII, as the PDK1 phosphorylation site on PAK1. Threonine 423 is a previously identified PAK1 autophosphorylation site that lies within a PAK consensus phosphorylation sequence. After pretreatment with phosphatases, autophosphorylation of PAK1 occurred at all major sites except threonine 423. A phosphothreonine 423-specific antibody detected phosphorylation of recombinant, catalytically inactive PAK1 after incubation with wild-type PAK1, indicating phosphorylation of threonine 423 occurs by an intermolecular mechanism. The biological significance of PDK1 phosphorylation of PAK1 at threonine 423 in vitro is supported by the observation that these two proteins interact in vivo and that PDK1-phosphorylated PAK1 has an increased activity toward substrate. An increase of phosphorylation of catalytically inactive PAK1 was observed in COS-7 cells expressing wild-type, but not catalytically inactive, PDK1 upon elevation of intracellular sphingosine levels. PDK1 phosphorylation of PAK1 was not blocked by pretreatment with wortmannin or when PDK1 was mutated to prevent phosphatidylinositol binding, indicating this process is independent of phosphatidylinositol 3-kinase activity. The data presented here provide evidence for a novel mechanism for PAK1 regulation and activation.