Primary human endothelial cells support direct but not antibody‐dependent enhancement of dengue viral infection

Microvascular plasma leakage is the hallmark of dengue hemorrhagic fever and dengue shock syndrome. The precise molecular mechanisms leading to microvascular leakage are yet to be determined, but dengue virus (DENV) infection and consequent endothelial cell death has been suggested as its major cause. However, the extent of endothelial cell permissiveness to DENV infection and the magnitude of cell death following DENV infection are controversial. To clarify this issue, we analyzed the kinetics and consequences of DENV infection of human umbilical vein endothelial cells (HUVEC) using a novel molecularly cloned DENV2‐16681 virus. Viral replication was detected as early as 24 hr post‐infection by RT‐PCR and plaque assays. However, merely 2% of HUVEC were DENV antigen‐positive even after 96 hr of infection as measured by the FACS indirect immunofluorescence assays. Unlike monocytes/macrophages, HUVEC did not support antibody dependent enhancement of dengue viral infection due to a lack of FcγRI and FcγRII. Furthermore, DENV infection did not increase HUVEC apoptosis as compared to mock‐infected cells. Because in vitro only a small percentage of endothelial cells were productively infected in vitro with no significant apoptosis occurring in either infected or bystander cells, it would be important to re‐examine whether direct dengue viral infection of endothelium is the major cause of the extensive vascular leakage observed in patients with dengue hemorrhagic fever and dengue shock syndrome. J. Med. Virol. 81:519–528, 2009. © 2009 Wiley‐Liss, Inc.     

Lymphatic tissue in Argentine hemorrhagic fever. Pathologic features

Spleen and lymph nodes from seven fatal cases of Argentine hemorrhagic fever were studied by light and electron microscopy and by immunofluorescent techniques. In addition, Junin virus infectivity was determined. Destruction of splenic white pulp and of lymph node cortex was observed. The ultrastructural study showed a characteristic cytopathic effect on the macrophages with presence of intracisternal virus-like particles and occasional virions budding from the plasma membrane. The immunofluorescent techniques indicated the presence of Junin virus antigenic determinants in the cytoplasm of macrophages in the examined organs. Junin virus titers were three times higher than in peripheral blood. These results suggest that lymphatic tissue is one of the main sites of viral replication and that the macrophages are the targets of Junin virus.     

Cross-protection in nonhuman primates against Argentine hemorrhagic fever.

The susceptibility of the marmoset Callithrix jacchus to Tacaribe virus infection was investigated to perform cross-protection studies between Junin and Tacaribe viruses. Five marmosets inoculated with Tacaribe virus failed to show any signs of disease, any alterations in erythrocyte, leukocyte, reticulocyte, and platelet counts or any changes in hematocrit or hemoglobin values. No Tacaribe virus could be recovered from blood at any time postinfection. Anti-Tacaribe neutralizing antibodies appeared 3 weeks postinfection. The five Tacaribe-infected marmosets and four noninfected controls were challenged with the pathogenic strain of Junin virus on day 60 post-Tacaribe infection. The former group showed no signs of disease, no viremia, and no challenge virus replication, whereas the control group exhibited the typical symptoms of Argentine hemorrhagic fever, high viremia, and viral titers in organs. Soon after challenge, the Tacaribe-protected marmosets synthesized neutralizing antibodies against Junin virus. These results indicate that the marmoset C. jacchus can be considered an experimental model for protection studies with arenaviruses and that the Tacaribe virus could be considered as a potential vaccine against Junin virus.     

Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression.

Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF.     

Evidence for two mechanisms of dengue virus infection of adherent human monocytes: trypsin-sensitive virus receptors and trypsin-resistant immune complex receptors.

Trypsin treatment of adherent human monocytes greatly reduced or eliminated the ability of these cells to support dengue virus replication. However, addition of dilute (nonneutralizing) antibody to the inoculum and the culture medium resulted in viral yields similar to those from monocytes not treated with trypsin. These results suggested that viral entry was facilitated by phagocytosis of immune complexes via Fc receptors on the monocytes. This concept was tested by (i) pretreating monocytes with aggregated gamma globulin, which resulted in a 40-fold reduction of viral yields after infection with dilute antibody-virus complexes and (ii) forming an immune complex with virus, antivirus F(ab')2 fragments, and rabbit anti-human Fab. Whereas F(ab')2 fragments alone would not enhance virus replication in trypsin-treated monocytes, the immune complex containing a rabbit Fc piece did increase the yield of dengue virus. These results suggest that dengue virus can infect a cultured monocyte in two ways: (i) through a viral receptor that is trypsin sensitive or (ii) through an Fc receptor that is not trypsin sensitive.     

Antibodies from dengue patient sera cross-react with endothelial cells and induce damage

Dengue virus infection causes a wide range of diseases from the mild febrile illness dengue fever to the life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage and hemorrhagic syndrome are the clinical features associated with dengue infection, yet the mechanisms remain unclear. In this study, the cross-reactivity of dengue patient sera with endothelial cells was demonstrated. There were higher percentages of endothelial cells reactive with dengue hemorrhagic fever/dengue shock syndrome patient sera than those with dengue fever patient sera. The percentages of endothelial cells reactive with patient serum IgM were higher than those with IgG. Further studies showed that the endothelial cell binding activity was inhibited by pretreatment with dengue virus nonstructural protein 1 (NS1). The antibodies against NS1 produced after dengue virus infection may, at least in part, account for the cross-reactivity of patient sera with endothelial cells. Furthermore, dengue patient sera induced endothelial cell apoptosis via a caspase-dependent pathway that was also inhibited by NS1 pretreatment. In addition to apoptosis, patient sera caused cell lysis in the presence of complement, and DHF/DSS patient sera showed higher percentages of cytotoxicity than dengue fever patient sera. Thus, the generation of cross-reactive autoantibodies against endothelial cells would lead to their dysfunction, which may play a role in the pathogenesis of dengue virus infection.     

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay

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.     

Pathogenesis of Ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells

Ebola virus (EBOV) infection causes a severe and often fatal hemorrhagic disease in humans and nonhuman primates. Whether infection of endothelial cells is central to the pathogenesis of EBOV hemorrhagic fever (HF) remains unknown. To clarify the role of endothelial cells in EBOV HF, we examined tissues of 21 EBOV-infected cynomolgus monkeys throughout time, and also evaluated EBOV infection of primary human umbilical vein endothelial cells and primary human lung-derived microvascular endothelial cells in vitro. Results showed that endothelial cells were not early cellular targets of EBOV in vivo, as viral replication was not consistently observed until day 5 after infection, a full day after the onset of disseminated intravascular coagulation. Moreover, the endothelium remained relatively intact even at terminal stages of disease. Although human umbilical vein endothelial cells and human lung-derived microvascular endothelial cells were highly permissive to EBOV replication, significant cytopathic effects were not observed. Analysis of host cell gene response at 24 to 144 hours after infection showed some evidence of endothelial cell activation, but changes were unremarkable considering the extent of viral replication. Together, these data suggest that coagulation abnormalities associated with EBOV HF are not the direct result of EBOV-induced cytolysis of endothelial cells, and are likely triggered by immune-mediated mechanisms.     

Dengue viruses can infect human primary lung epithelia as well as lung carcinoma cells, and can also induce the secretion of IL-6 and RANTES

Dengue viruses (DENV) are herein demonstrated for the first time as being able to infect and replicate in human primary lung epithelium and various lung cancer cell lines. The detection of dengue virus particles and viral negative strand RNA synthesis in the cell, in conjunction with the release of viral progenies in culture supernatants, support the notion that lung cells are susceptible to dengue virus infection. The replication efficiency of DENV in lung cancer cells from high to low is: DEN-2 (dengue virus type-2), DEN-3, DEN-4 and DEN-1. Moreover, the susceptibility of the six lung cancer cell lines to DEN-2 infection is: SW1573>A549>H1435; H23; H520; Bes2B. DEN-2 infection significantly increased the expression levels of IL-6 and RANTES in four of the six lung cancer cell lines, which is consistent with the high expression levels of these molecules in DHF/DSS patients. IL-6 expression induced by DEN-2 infection was NF-kappaB dependent. In summary, our results indicate that lung epithelial cell is a possible target of dengue viruses and IL-6 and RANTES may play pivotal roles in lung related immuno-pathogenesis.     

FasL/Fas pathway is involved in dengue virus induced apoptosis of the vascular endothelial cells

The hallmark of the dengue hemorrhagic fever/dengue shock syndrome is hematologic abnormality. The pathogenesis of dengue hemorrhagic fever/dengue shock syndrome remains unknown. Our work showed that the dengue virus serotype‐2 induced apoptosis in human umbilical vein endothelial cells. Fas (CD95), Tumor Necrosis Factor receptors, and Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors are the most common death receptors, which can induce apoptosis. Compared with the untreated human umbilical vein endothelial cells, Fas expression was increased both in the mRNA level and on the surface of infected human umbilical vein endothelial cells. FasL was expressed at similar levels on human umbilical vein endothelial cells over a course of dengue virus serotype‐2 infection, but the expression in mRNA level was increased in infected human umbilical vein endothelial cells. It is possible that there is soluble FasL secreted from human umbilical vein endothelial cells in the supernatant. Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptor 1 and Tumor Necrosis Factor receptors 1–2 were constantly very low, whereas Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors 2–4 decreased after dengue virus serotype‐2 infection. This result suggested that dengue virus serotype‐2 may inhibit Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors‐induced apoptosis. The apoptotic rates in human umbilical vein endothelial cells were decreased upon the addition of caspase family inhibitors. In addition, activated caspase 8 and caspase 3 were also observed by Western blot following dengue virus serotype‐2 infection. Thus, it is shown that the Fas/FasL pathway may participate in dengue virus‐induced apoptosis of vascular endothelial cells in vitro. J. Med. Virol. 82:1392–1399, 2010. © 2010 Wiley‐Liss, Inc.     

Phenotypic and genotypic characterization of dengue virus isolates differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome

Dengue viruses (DENV) cause 50-100 million cases of acute febrile disease every year, including 500,000 reported cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Viral factors have been proposed to influence the severity of the disease, but markers of virulence have never been identified on DENV. Three DENV serotype-1 isolates from the 2007 epidemic in Cambodia that are derived from patients experiencing the various clinical forms of dengue were characterized both phenotypically and genetically. Phenotypic characteristics in vitro, based on replication kinetics in different cell lines and apoptosis response, grouped isolates from DF and DHF patients together, whereas the virus isolate from a DSS patient showed unique features: a lower level of replication in mammalian cells and extensive apoptosis in mosquito cells. Genomic comparison of viruses revealed six unique amino acid residues in the membrane, envelope, and in non-structural genes in the virus isolated from the DSS patient.     

Treatment of junin virus-infected guinea pigs with immune serum: development of late neurological disease

Guinea pigs infected with Argentine hemorrhagic fever virus (Junin) were treated with pooled, homologous convalescent sera. Use of 15,000 or 5,000 therapeutic units of immune sera prevented all signs of illness when administered within 24 hr of infection. We could also prevent illness and death in infected guinea pigs as late as 6 days after infection if we used more antisera (30,000 therapeutic units/kg). In some treatment groups, surviving animals developed a late neurological syndrome with prominent rear-limb paralysis. Treated animals typically expressed higher viral titers in the brain than in any other organ. There appeared to be no acute exacerbation of disease by antibody administration. Our data suggest that, after replicating peripherally, Junin virus infects the brain where circulating immunoglobulins may not eliminate viable virus. Subsequent replication of virus in the brain may generate a neurological phase of the illness. Histological examination of brains from guinea pigs in treatment groups favoring the neurological phase of illness showed encephalitis, meningitis, and swollen astrocytes, suggestive of neuronal degeneration. There is likely a delicate balance among presence of virus in the brain, the amount of antibody transported into the central nervous system, and the occurrence of this late neurological aspect of experimental Argentine hemorrhagic fever. Further study of this model may elucidate factors relevant in understanding the continuing problem of the late neurological syndrome seen in some human cases of Argentine hemorrhagic fever treated with immune plasma.     

Dengue virus replication in a polyploid mosquito cell culture grown in serum-free medium

A subline of a polyploid cell line (TRA-284) derived from a nonbiting mosquito, Toxorhynchites amboinensis, was adapted to a serum-free medium. The sensitivity of the subline (TRA-284-SF) to all serotypes of adapted dengue viruses was generally comparable to that of Aedes albopictus (C6/36), and DEN 3 viruses replicated to higher titers in TRA-28F-SF cells than in C6/36 cells. The subline was found to be useful for isolation of dengue viruses from human serum, since isolation rates were higher in TRA-284-SF cells than in C6/36 cells. The advantages of using a serum-free medium and mosquito cells for virus isolation are discussed.     

Mumps virus replication in human lymphoid cell lines and in peripheral blood lymphocytes: preference for T cells.

The replication of mumps virus was studied in human continuous lymphoblastoid cell lines (LCLs) with T or B characteristics and in lymphocyte subpopulations derived from peripheral blood. T-LCLs supported effective virus replication as shown by high titers of free and cell-associated virus over 1 to 4 days after infection. By immunofluorescence analysis, the majority of cells were positive for mumps virus antigens. In contrast, the B-cell lines produced low titers of infectious virus, and only a small percentage expressed viral antigens. This resistance of the B-LCLs was found with several mumps virus strains. Cultures of peripheral blood mononuclear cells also supported mumps virus replication. Very high titers of infectious virus (10(8) PFU/ml) were observed in cultures prestimulated with phytohaemagglutinin. Studies with enriched T and B cells point to the activated T lymphocyte as the major virus-producing cell.     

SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium

SARS coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The severe and sudden onset of symptoms, resulting in an atypical pneumonia with dry cough and persistent high fever in cases of severe acute respiratory virus brought to light the importance of coronaviruses as potentially lethal human pathogens and the identification of several zoonotic reservoirs has made the reemergence of new strains and future epidemics all the more possible. In this chapter, we describe the pathology of SARS-CoV infection in humans and explore the use of two models of the human conducting airway to develop a better understanding of the replication and pathogenesis of SARS-CoV in relevant in vitro systems. The first culture model is a human bronchial epithelial cell line Calu-3 that can be inoculated by viruses either as a non-polarized monolayer of cells or polarized cells with tight junctions and microvilli. The second model system, derived from primary cells isolated from human airway epithelium and grown on Transwells, form a pseudostratified mucociliary epithelium that recapitulates the morphological and physiological features of the human conducting airway in vivo. Experimental results using these lung epithelial cell models demonstrate that in contrast to the pathology reported in late stage cases SARS-CoV replicates to high titers in epithelial cells of the conducting airway. The SARS-CoV receptor, human angiotensin 1 converting enzyme 2 (hACE2), was detected exclusively on the apical surface of cells in polarized Calu-3 cells and human airway epithelial cultures (HAE), indicating that hACE2 was accessible by SARS-CoV after lumenal airway delivery. Furthermore, in HAE, hACE2 was exclusively localized to ciliated airway epithelial cells. In support of the hACE2 localization data, the most productive route of inoculation and progeny virion egress in both polarized Calu-3 and ciliated cells of HAE was the apical surface suggesting mechanisms to release large quantities of virus into the lumen of the human lung. Preincubation of the apical surface of cultures with antisera directed against hACE2 reduced viral titers by two logs while antisera against DC-SIGN/DC-SIGNR did not reduce viral replication levels suggesting that hACE2 is the primary receptor for entry of SARS-CoV into the ciliated cells of HAE cultures. To assess infectivity in ciliated airway cultures derived from susceptible animal species we generated a recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF 7a/7b) and insertion of the green fluorescent protein (GFP) resulting in SARS-CoV GFP. SARS-CoV GFP replicated to similar titers as wild type viruses in Vero E6, MA104, and CaCo2 cells. In addition, SARS-CoV replication in airway epithelial cultures generated from Golden Syrian hamster tracheas reached similar titers to the human cultures by 72 h post-infection. Efficient SARS-CoV infection of ciliated cell-types in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.     

H1N1亚型流感病毒在A549和BEAS-2B细胞中复制情况的初步研究

目的 探讨不同宿主来源的H1N1亚型流感病毒在A549和BEAS-2B细胞的复制情况.方法 用分离自人、禽、猪三种宿主的7株H1N1甲型流感病毒分别接种A549和BEAS-2B细胞,分析病毒感染细胞后不同时段的特点;应用受体类型不同的红细胞进行微量血凝试验,检测流感病毒的受体结合特性;同时检测了A549和BEAS-2B细胞表面的受体分布情况.结果 三种宿主来源的H1N1亚型流感病毒感染A549细胞,24 h后CPE十分明显,36 h病毒滴度达到最高值;而感染BEAS-2B细胞后,从24 h-120 h CPE都不是很明显,且所有病毒的病毒滴度都很低.对6株H1N1流感病毒的受体结合特性进行了筛查,发现部分测试病毒具有SA a-2,6Gal受体结合特异性.而A549和BEAS-2B细胞表面均含有SA a-2,3Gal及SA a-2,6Gal受体,且A549细胞表面糖受体含量明显高于BEAS-2B细胞.结论 不同宿主来源的H1N1亚型流感病毒对A549细胞都易感并能有效增殖复制,而对具有相似受体特性、上皮组织来源的BEAS-2B细胞不易感,提示支持流感病毒有效感染、复制存在宿主内的调节机制.     

Senescence Affects Endothelial Cells Susceptibility to Dengue Virus Infection

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.