Irene Bosch

Dengue Virus Induces Novel Changes in Gene Expression of Human Umbilical Vein Endothelial Cells

ABSTRACT Endothelial cells are permissive to dengue virus (DV) infection in vitro, although their importance as targets of DV infection in vivo remains a subject of debate. To analyze the virus-host interaction, we studied the effect of DV infection on gene expression in human umbilical vein endothelial cells (HUVECs) by using differential display reverse transcription-PCR (DD-RTPCR), quantitative RT-PCR, and Affymetrix oligonucleotide microarrays. DD identified eight differentially expressed cDNAs, including inhibitor of apoptosis-1, 2′-5′ oligoadenylate synthetase (OAS), a 2′-5′ OAS-like (OASL) gene, galectin-9, myxovirus protein A (MxA), regulator of G-protein signaling, endothelial and smooth muscle cell-derived neuropilin-like protein, and phospholipid scramblase 1. Microarray analysis of 22,000 human genes confirmed these findings and identified an additional 269 genes that were induced and 126 that were repressed more than fourfold after DV infection. Broad functional responses that were activated included the stress, defense, immune, cell adhesion, wounding, inflammatory, and antiviral pathways. These changes in gene expression were seen after infection of HUVECs with either laboratory-adapted virus or with virus isolated directly from plasma of DV-infected patients. Tumor necrosis factor alpha, OASL, and MxA and h-IAP1 genes were induced within the first 8 to 12 h after infection, suggesting a direct effect of DV infection. These global analyses of DV effects on cellular gene expression identify potentially novel mechanisms involved in dengue disease manifestations such as hemostatic disturbance.

Increased Production of Interleukin-8 in Primary Human Monocytes and in Human Epithelial and Endothelial Cell Lines after Dengue Virus Challenge

ABSTRACT The more severe form of dengue virus infection, dengue hemorrhagic fever, is characterized by plasma leakage and derangements in hemostasis. As elevated interleukin-8 (IL-8) levels have been observed in sera from patients with more severe disease manifestations, a study was initiated to look at the effect of dengue virus infection in vitro on proinflammatory cytokine secretion and expression. A significant increase in IL-8 levels in the culture supernatant of primary human monocytes infected with dengue 2 virus (D2V) New Guinea C (NGC) was found by enzyme-linked immunosorbent assay. Additionally, by reverse transcriptase PCR, the mRNA was also augmented. Among the proinflammatory cytokines and their mRNAs measured (IL-6, IL-1β, IL-8, and tumor necrosis factor alpha), IL-8 showed the greatest change following D2V infection. Similarly, two cell lines, 293T (a human epithelial cell line) and ECV304 (an endothelial cell line), were permissive to D2V NGC and responded to the infection by increasing the synthesis of IL-8. Nuclear factor kappa B (NF-κB) and nuclear factor IL-6 (NFIL-6) are primary mediators of IL-8 expression. We studied the transcriptional regulation of IL-8 in the ECV304 and 293T cell lines and found that the induction of IL-8 gene expression involved the activation of NF-κB (P = 0.001) and, to a lesser extent, the activation of NFIL-6 in ECV304 cells only. We next observed by the chromatin immunoprecipitation procedure in vivo acetylation of core histones bound to the IL-8 promoter after D2V infection. IL-8 produced by infected monocytes and also IL-8 that may be produced by endothelial or other epithelial cells is associated with the hyperacetylation of histones bound to the IL-8 promoter in addition to the activation of transcription by NF-κB. We hypothesize that the overall increase in IL-8 synthesis observed in this in vitro study may play a role in the pathogenesis of the plasma leakage seen in dengue hemorrhagic fever and dengue shock syndrome.

Gene expression signature in organized and growth-arrested mammary acini predicts good outcome in breast cancer.

Nonmalignant human mammary epithelial cells (HMEC) seeded in laminin-rich extracellular matrix (lrECM) form polarized acini and, in doing so, transit from a disorganized proliferating state to an organized growth-arrested state. We hypothesized that the gene expression pattern of organized and growth-arrested HMECs would share similarities with breast tumors with good prognoses. Using Affymetrix HG-U133A microarrays, we analyzed the expression of 22,283 gene transcripts in 184 (finite life span) and HMT3522 S1 (immortal nonmalignant) HMECs on successive days after seeding in a lrECM assay. Both HMECs underwent growth arrest in G0-G1 and differentiated into polarized acini between days 5 and 7. We identified gene expression changes with the same temporal pattern in both lines and examined the expression of these genes in a previously published panel of microarray data for 295 breast cancer samples. We show that genes that are significantly lower in the organized, growth-arrested HMEC than in their proliferating counterparts can be used to classify breast cancer patients into poor and good prognosis groups with high accuracy. This study represents a novel unsupervised approach to identifying breast cancer markers that may be of use clinically.

Heart and skeletal muscle are targets of dengue virus infection.

Background: Dengue fever is one of the most significant re-emerging tropical diseases, despite our expanding knowledge of the disease, viral tropism is still not known to target heart tissues or muscle. Methods: A prospective pediatric clinical cohort of 102 dengue hemorrhagic fever patients from Colombia, South America, was followed for 1 year. Clinical diagnosis of myocarditis was routinely performed. Electrocardiograph and echocardiograph analysis were performed to confirm those cases. Immunohistochemistry for detection of dengue virus and inflammatory markers was performed on autopsied heart tissue. In vitro studies of human striated skeletal fibers (myotubes) infected with dengue virus were used as a model for myocyte infection. Measurements of intracellular Ca2+ concentration as well as immunodetection of dengue virus and inflammation markers in infected myotubes were performed. Results: Eleven children with dengue hemorrhagic fever presented with symptoms of myocarditis. Widespread viral infection of the heart, myocardial endothelium, and cardiomyocytes, accompanied by inflammation was observed in 1 fatal case. Immunofluorescence confocal microscopy showed that myotubes were infected by dengue virus and had increased expression of the inflammatory genes and protein IP-10. The infected myotubes also had increases in intracellular Ca2+ concentration. Conclusions: Vigorous infection of heart tissues in vivo and striated skeletal cells in vitro are demonstrated. Derangements of Ca2+ storage in the infected cells may directly contribute to the presentation of myocarditis in pediatric patients.

Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells

Targeted gene transfer by nonviral vectors can be achieved through incorporation of specific ligand(s) into the vectors. In this study, the effects of incorporation of an anti-ErbB2 single-chain antibody fragment (ScFv) into nonviral vectors for targeted gene delivery were investigated. The ML39 ScFv, selected from a human ScFv phage display library and affinity matured in vitro ( K d=1×10−9 M), was used as ligand specific for the extracellular domain of the tumor surface protein, ErbB2. Two approaches were taken: (a) development of a vector that is composed of a bifunctional fusion protein capable of binding DNA with the ErbB2-specific ML39 ScFv at its N-terminus and a truncated form of human protamine at its C-terminus, and (b) formulation and evaluation of delivery vectors consisting of three independent components including ML39 ScFv, protamine, and cationic lipids. We demonstrate that fusion proteins comprised of the ML39 ScFv and a truncated form of protamine, denoted as ScFv-P-S, can selectively deliver exogenous DNA into ErbB2(+) cells, with an 8- to 10-fold increase in expression levels of the luciferase reporter gene in ErbB2(+) cells as compared to ErbB2(−) cells. In addition, vectors formulated by appropriately mixing DNA, ScFv, protamine, and lipids in vitro could even more efficiently deliver the reporter gene into ErbB2(+) cells with approximately 5-fold increase in gene expression in ErbB2(+) cell as compared to ErbB2(−) cells. Expression and refolding of the ScFv fusion proteins, in addition to determination of optimal conditions for vector development using these approaches, are discussed. Cancer Gene Therapy (2001) 8, 555–565

TRAIL Is a Novel Antiviral Protein against Dengue Virus

ABSTRACT Dengue fever is an important tropical illness for which there is currently no virus-specific treatment. To shed light on mechanisms involved in the cellular response to dengue virus (DV), we assessed gene expression changes, using Affymetrix GeneChips (HG-U133A), of infected primary human cells and identified changes common to all cells. The common response genes included a set of 23 genes significantly induced upon DV infection of human umbilical vein endothelial cells (HUVECs), dendritic cells (DCs), monocytes, and B cells (analysis of variance, P < 0.05). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the common response genes, was identified as a key link between type I and type II interferon response genes. We found that DV induces TRAIL expression in immune cells and HUVECs at the mRNA and protein levels. The induction of TRAIL expression by DV was found to be dependent on an intact type I interferon signaling pathway. A significant increase in DV RNA accumulation was observed in anti-TRAIL antibody-treated monocytes, B cells, and HUVECs, and, conversely, a decrease in DV RNA was seen in recombinant TRAIL-treated monocytes. Furthermore, recombinant TRAIL inhibited DV titers in DV-infected DCs by an apoptosis-independent mechanism. These data suggest that TRAIL plays an important role in the antiviral response to DV infection and is a candidate for antiviral interventions against DV.

West Nile Virus, Venezuela

To the Editor: West Nile virus (WNV; genus Flavivirus; family Flaviviridae) has been perpetuating in North America since 1999 (1). However, its status as a self-perpetuating pathogen in South America remains uncertain. Infected horses and birds have been reported in various Caribbean Islands, Mexico, and northern Central America (2,3). In South America, isolated reports of infected dead-end hosts (horses) have come from northern Colombia and Argentina but they lack evidence for infection in avian amplifying hosts (4,5). We report serologic evidence of establishment of WNV in South America. Serum samples from birds and horses from 33 locations in Venezuela (Appendix Table) were screened for immunoglobulin G (IgG) antibodies against WNV antigen by ELISA (6) and confirmed by plaque reduction neutralization test (PRNT) as previously described (7). The flavivirus generating the IgG response was identified by using the following criteria: 90% inhibition of virus in serum diluted at least 1:40 and 4-fold greater neutralizing antibody titer compared with closely related flaviviruses. IgG antibody against flavivirus was detected by ELISA in 14 of 576 resident birds, including 5 Turdus leucomelas, 3 Gallus gallus (captive), 2 Campylorhamphus trochilirostris, and 1 each of Elaenia flavogaster, Coereba flaveola, Thraupis palmarum, and Anisognathus flavinucha. WNV was confirmed as the etiologic agent of infection in 5 adult birds (3 T. leucomelas [pale-breasted thrush], 1 C. flaveola [bananaquit], and 1 G. gallus [domestic chicken] with the earliest collection date in February 2006); virus neutralization titers ranged from 80 to 320. One serum sample cross-reacted with other flaviviruses tested, with equivalent titers to WNV, Saint Louis encephalitis virus (SLEV), and Ilheus virus (ILHV) and was thus considered infected with an undetermined flavivirus. Seven serum samples were negative (antibody titers <20), and 1 sample was not tested because of insufficient sample volume. Antibody against flavivirus was detected by ELISA in 141 of 791 horses, and 34 (4.3%) were confirmed positive for WNV infection by PRNT; viral titers ≥640 occurred in half of these horses. The earliest collection date for a WNV-positive horse was February 2004 and the most recent was May 2006. Specific WNV-reactive equine serum samples were distributed in valley regions (prevalence 1.3%), savannah grasslands (2.4%), the western region of Zulia (0.4%) and the Central Lake Basin (0.3%). A total of 46 (5.8%) equine serum samples were positive for neutralizing antibody to SLEV, and 8 (1.0%) samples were positive for neutralizing antibodies to ILHV. Forty-nine samples neutralized at least 2 of the 3 viruses and were classified as undetermined flaviviruses. Serum samples from 2 horses were negative in neutralization assays; 2 others were not tested because of insufficient sample volume. WNV-infected resident birds, rather than an importation event, are the basis of establishment of WNV in South America. We hypothesize that ornithophilic mosquitoes (such as some Culex spp.), which are present in the area in consistently high numbers, acquired the virus through hematophagous feeding on recently infected, migrating birds. Once introduced to local mosquitoes, virus is amplified among susceptible resident birds fed upon by ornithophilic mosquitoes. This pattern allows perpetuation and subsequent establishment of virus in a continuous transmission cycle, as opposed to infection of dead-end hosts, e.g., horses. This is the first report of WNV infection in South American birds and definitive establishment of the virus in South America. We observed varying WNV seroprevalence rates in birds and horses across regions in Venezuela (Figure). These differences reflect the focal and stochastic nature of arbovirus transmission, which depends upon many ecologic factors. One possible explanation for the greater seroprevalence in the central and eastern llanos (savannahs) and valley regions, compared with the coastal western region of Zulia State (p<0.0001, by Pearson’s χ2 test) would be virus introduction by migrating birds by an eastern migration route. Figure Collection sites for West Nile virus (WNV) in Venezuela. Symbols represent results of tests for specific antibodies to WNV in serum samples of birds and horses (viral titers in a 90% plaque reduction neutralization test >40 and a 4-fold differential ... Existence of several closely related flaviviruses in the American tropics (8–10) may convey cross-protection in animals (e.g., ILHV and SLEV) or humans (dengue viruses, yellow fever virus), thereby potentially diminishing disease caused by a newly introduced flavivirus such as WNV. Although ILHV infection has not been detected in Venezuela, this flavivirus is prevalent in Brazil, Peru, French Guyana, Trinidad, and Colombia. Our study demonstrated widespread distribution of ILHV in Venezuela. Other South American flaviviruses, such as Bussuquara, Cacipacore, and Iguape, and as yet undiscovered viruses may also circulate in Venezuela. We encourage those involved in the public and animal health systems in Venezuela to consider zoonotic flaviviruses in the differential diagnoses of human and equine cases of encephalitis and to consider ecologic surveillance for zoonotic flaviviruses in mosquito and vertebrate host populations. We recommend monitoring blood and organ donations for flavivirus infections. Our study sheds light on flavivirus distribution in Venezuela. However, nothing else is known about the ecology of zoonotic flaviviruses in this country. Such knowledge will be essential for designing effective surveillance and control should these viruses be shown to cause human illnesses.

Zika virus evolution and spread in the Americas

Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests.

Elevated levels of soluble ST2 protein in dengue virus infected patients

Levels of the soluble form of the interleukin-1 receptor-like 1 protein (IL-1RL-1/ST2) are elevated in the serum of patients with diseases characterized by an inflammatory response. The objective of this study was to determine the concentration of soluble ST2 (sST2) in dengue infected patients during the course of the disease. Twenty-four patients with confirmed dengue infection, classified as dengue fever, and 11 patients with other febrile illness (OFI) were evaluated. Levels of sST2 in serum and laboratory variables usually altered during dengue infections were measured. Dengue infected patients had higher serum sST2 levels than OFI at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004, respectively). Patients with secondary dengue infections had higher serum sST2 levels compared with patients with primary dengue infections (p=0.047 at last day of fever and p=0.030 at defervescence). Furthermore, in dengue infected patients, we found a significant negative correlation of sST2 with platelet and WBC counts, and positive correlation with thrombin time and transaminases activity. We suggest that sST2 could be a potential marker of dengue infection, could be associated with severity or could play a role in the immune response in secondary dengue virus infection.

CD8+ T Cells Use TRAIL To Restrict West Nile Virus Pathogenesis by Controlling Infection in Neurons

ABSTRACT Previous studies of mice have demonstrated that an orchestrated sequence of innate and adaptive immune responses is required to control West Nile virus (WNV) infection in peripheral and central nervous system (CNS) tissues. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; also known as CD253) has been reported to inhibit infection with dengue virus, a closely related flavivirus, in cell culture. To determine the physiological function of TRAIL in the context of flavivirus infection, we compared the pathogenesis of WNV in wild-type and TRAIL −/− mice. Mice lacking TRAIL showed increased vulnerability and death after subcutaneous WNV infection. Although no difference in viral burden was detected in peripheral tissues, greater viral infection was detected in the brain and spinal cord at late times after infection, and this was associated with delayed viral clearance in the few surviving TRAIL −/− mice. While priming of adaptive B and T cell responses and trafficking of immune and antigen-specific cells to the brain were undistinguishable from those in normal mice, in TRAIL −/− mice, CD8+ T cells showed qualitative defects in the ability to clear WNV infection. Adoptive transfer of WNV-primed wild-type but not TRAIL −/− CD8+ T cells to recipient CD8−/− mice efficiently limited infection in the brain and spinal cord, and analogous results were obtained when wild-type or TRAIL −/− CD8+ T cells were added to WNV-infected primary cortical neuron cultures ex vivo. Collectively, our results suggest that TRAIL produced by CD8+ T cells contributes to disease resolution by helping to clear WNV infection from neurons in the central nervous system.