Mechanism Study Of Dengue Virus Type3Infecting The Human Brain Microvascular Endothelial Cells

1. Background and ObjectiveDengue is the most important arthropod-borne human viral disease. Estimates of the annual number of dengue fever (DF) cases in the world vary widely, ranging from70to500million, including24,000deaths. It is caused by dengue virus (DENV), a single stranded RNA virus that belongs to the Flaviviridae family. DENV infection may be asymptomatic or cause a mild self-limited fever, dengue fever (DF), but may also evolve to the severe and life-threatening diseases known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The main symptoms of DHF/DSS are the loss of intravascular fluid volume, with consequent raised hematocrit, hypotension and serous effusions, which point to alterations in the vascular endothelium as a major event in the disease progression.Dengue has been regarded as a nonneurotropic virus. The neurological spectrum of dengue patients has been limited because of small number of case reports. The association of dengue infection with unusual neurological manifestations was first reported by Sanguansermsri et al in1976. Since that time, there have been reports describing neurological involvement in dengue virus infection. The exact incidence of various neurological complications is uncertain. The reported incidence of encephalopathy and encephalitis, the most common neurological complications of dengue, has been found to vary between0.5%and6.2%. However, the proportional positivity was higher for the group of patients who died than for those who recovered from dengue infection. The frequency of CNS infection by DENV in dengue cases with fatal outcomes during a dengue epidemic period even up to48.8%in the State of Ceara’, northeastern Brazil. Also, recent observations indicate that the clinical profile of dengue is changing, and that neurological manifestations are being reported more frequently.Neurologic involvement in dengue infections has been referred to as encephalopathy and attributed to fluid extravasations, cerebral edema, hyponatremia, liver or renal failure, and not to encephalitis due to localized invasion of the CNS. However, an autopsy study of a child with dengue encephalitis has shown histological evidence of encephalitis. Dengue antigen has been detected in the brain. Dengue virus was isolated in the cerebrospinal fluid (CSF) of patients with dengue encephalitis and has been detected by immunehistochemistry and by reverse transcription polymerase chain reaction (RT-PCR) in the inferior olivery nucleus and granular layer of cerebellum. Immunoreactivity was observed in neurons, microglia and endothelial cells. These providing strong evidence that DENV has neurovirulent properties.It is known that the Blood Brain Barrier (BBB) is a unique membranous structure in brain capillaries that tightly segregates the brain from systemic blood circulation. The BBB can protect the brain from the intrusion of harmful substances. Three elements underlie the BBB function:(1) a physical barrier comprised of tight junctions (TJs), which form a tight seal to intercellular diffusion,(2) the cells themselves, which exhibit a low rate of endocy-tosis, and (3) a metabolic barrier, consisting of specific membrane transporters expressed by endothelial cells. Despite this barrier, some viruses such as Dengue virus, West Nile virus and Eastern equine encephalitis virus manage to gain access to the central nervous system (CNS) through mechanisms that are largely unknown.Since BBB endothelial cells are directly exposed to cell-free virus in the peripheral blood, DENV infection and replication in human brain microvascular endothelial cells (HBMEC), an important component of the BBB, can be one of the possible route by which DENV enters the CNS. Infection of BBB endothelial cells and changes in its properties as a result of virus infection have been shown for several viruses, specifically those which manifest acute neurodegeneration and neuroinflammation, such as simian immunodeficiency virus (SIV), measles virus and human T-cell leukemia virus. Also, cell-free DENV may cross the BBB either by transcytosis mechanism without altering the BBB integrity, or by modulating TJP of the BBB thereby allowing passage of not only cell-free virus, but also cell-associated virus, into the CNS. HIV induced changes in the brain microvascular cells, including alterations in the expressions of TJP and cell adhesion molecules (CAM), are associated with increased in flux of infected inflammatory cells as’Trojan Horse’and higher viral load in the CNS. Leukocyte adhesion and trafficking across endothelial cells depends on sequential activation and expression of cell surface adhesion molecules such as VCAM-1and ICAM-1in HIV and SIV infected BBB models. Moreover, Animal studies have shown that the DENV virus is known to release cytokines such as IFN-gamma, IL-6and MCP-1that could breach the blood-brain barrier, thus being capable of CNS invasion. This study characterizes DENV infection in HBMEC cells, specifically the effect on TJP and CAM expressions. Also, the production of cytokines in cells supernatant has been tested.2. Methods(1) DV3infection and replication kinetics in HBMECPost-infected C6/36cells were cultured in28℃incubator. Viruses were harvested after CPE over80%. Virus virulence was performed by cells culture titration. HBMEC were infected with D3at the multiplicity of infection (MOI) of5.The CPE of HBMEC was observed. Cells and supernatant were harvested at various time points(24h,48h,72h,96h). Taqman probe qRT-PCR was performed to test the fold-change of vrius copies in supernatant. The change of expression of non-structural protein1(NS1) at each time point was determined by immunofluorescence compared to corresponding uninfected control.(2)Fold-change in expressions of key tight junction proteins (TJP) in DV3infected HBMEC. The Primer Premier5.0and Oligo7.0software were used to design and evaluate the upstream and downstream primers of key TJP gene(ZO-1, Claudin-1, Occludin-1).Cellular RNA from mock-infected, MOI-5DV3-infected cells at different time points after infection were extracted. qRT-PCR with SYBEGEEN was performed to test the fold-change of TJP mRNA expression at each time point compared to corresponding un-infected control. Total cellular protein was extracted with RIPA from mock-infected and MOI-5DV3-infected cells from days1to4after infection. The change of expression of TJP at each time point was determined by Western Blot.(3)Fold-change in expressions of cell adhesion molecules (CAM) in DV3infected HBMEC.The Primer Premier5.0and Oligo7.0software were used to design and evaluate the upstream and downstream primers of cell adhesion molecules (CAM) gene(RANTES, E-selectin, PECAM, VCAM-1, ICAM-3). Cellular RNA from mock-infected, MOI-5WNV-infected cells at different time points after infection was extracted. qRT-PCR with SYBEGEEN was performed to test the fold-change of CAM mRNA expression at each time point compared to corresponding un-infected control. Total cellular protein was extracted with RIPA from mock-infected and MOI-5WNV-infected cells from days1to4after infection. The change of expression of CAM at each time point was determined by Western Blot.(4) Fold-change in expressions of cytokines in DV3infected HBMEC.HBMEC were infected with DV3at the multiplicity of infection (MOI) of5. Supernatant were harvested at various time points(24h,48h,72h).The fold-change of23cytokines expression in Supernatant at each time point compared to corresponding un-infected control were detected by Protein chip which performed by enzyme-linked immunosorbent assay. Significant increased cytokines in Protein chip was further validated using western blot (WB) and densitometric analysis.3. Result(1) DV3infection and replication kinetics in HBMECDV3can infect and replicate in HBMEC cells. According to the morphological observation,DV3can result in CPE of HBMEC which manifests with cells narrow, round, and fall off from the culture bottle and could became more severe from days1to5after infection.DV3replication was quantitated using qRT-PCR in DV3-infected cell supernatants collected at days1to4after infection. Productive DV3replication, in the form of release of virions was first detected at day1and gradually increased after infection in MOI-5infected cells. By using immunofluorescence microscopy, DV3antigen NS1was detected as early as24h after infection and the number of infected cells gradually increased.(2) Fold-change in expressions of key tight junction proteins (TJP) in DV3infected HBMEC.The expression of ZO-1and Claudin-1gene sharp increased from day1after MOI-5infection and decline to almost normal at days2after infection but not in cells un-infected. Significant increase in the ZO-1and claudin-1transcript level at day1after infection was further validated using western blot (WB) and densitometric analysis, and the increases of both of them were statistically significant. However, the marginal increase in Occludin-1mRNA expression was not observed using WB assay, suggesting that the mRNA increase was not significant.(3) Fold-change in expressions of cell adhesion molecules (CAM) in DV3infected HBMEC.The expression of VCAM-1gene sharp increased from day2after MOI-5infection and sustained higher than cells un-infected. RANTES was almost normal at first2days, then an sharp increase at day3. Significant increase in the RANTES and VCAM-1transcript level at day2after infection was further validated using western blot (WB) and densitometric analysis, and this increase of RANTES were statistically significant. However, the increase in VCAM-1mRNA expression was not observed using WB assay, suggesting that the mRNA increase was not significant.(4) Fold-change in expressions of cytokines in DV3infected HBMEC.The expression of GM-CSF、GRO-α MCP-2、MIG、RANTES show over1.5fold-change from day1after MOI-5infection and remained significantly high at days2and3after infection compare to cells un-infected. These were further validated using western blot (WB) and densitometric analysis.4. Conclusion(1) In this study we chose to investigate HBMEC cells because they cover the largest surface area of the BBB and therefore allow extensive targeting by neurotropic viruses.Our study demonstrating intracellular DV3replication by qRT-PCR, DV3antigen by immunocytochemistry and release of infectious virions by cell culture. This trend of virus replication is like other susceptible cells such as mononuclear or BHK-21cells where persistently virus replication is observed after infection, accompanied by CPE.AS critical players in the maintenance of BBB integrity, the infection HBMEC cells may be an important path for DV3enter BBB via the transcellular pathway.(2) Our results demonstrating significant up-regulation of ZO-1and claudin-1at transcriptional level. Down-regulation of ZO-1in microvascular endothelial cells has been observed at follow days in WB. It seems likely that it is a response of inflammatory mediators such as cytokines released by DV3-infected HBMEC cells. The change of TJP can be one of the mechanism by which DV3gains entry into the CNS via the paracellular pathway, with compromising the BBB integrity.(3) Infected cells assist in the entry of virus by’Trojan horse’mechanism, where infected monocytes/macrophages take up residence in the CNS and disseminate virus to neighboring brain cells. Significant up-regulation of cell adhesion molecules (CAM) such as RANTES was obsevered in our vitro study, which suggests that ’Trojan horse’mechanism may contributing to the severe inflammation observed in DV-associated meningitis.(4) The expression of GM-CSF, IL-13, MCP-1, MCP-2, MIG, RANTES show significant up-regulation in supernatant at various time points.These cytokines have been confirmed significantly related to chemotaxis, generation and maturation of inflammatory cells which were susceptible to DV. This suggests that DV3infection could promote local inflammatory response in BBB. These were further validated in follow study.