A new permanent cell line derived from the bank vole (Myodes glareolus) as cell culture model for zoonotic viruses

<p>Abstract</p> <p>Background</p> <p>Approximately 60% of emerging viruses are of zoonotic origin, with three-fourths derived from wild animals. Many of these zoonotic diseases are transmitted by rodents with important information about their reservoir dynamics and pathogenesis missing. One main reason for the gap in our knowledge is the lack of adequate cell culture systems as models for the investigation of rodent-borne (robo) viruses <it>in vitro</it>. Therefore we established and characterized a new cell line, BVK168, using the kidney of a bank vole, <it>Myodes glareolus, </it>the most abundant member of the <it>Arvicolinae </it>trapped in Germany.</p> <p>Results</p> <p>BVK168 proved to be of epithelial morphology expressing tight junctions as well as adherence junction proteins. The BVK168 cells were analyzed for their infectability by several arbo- and robo-viruses: Vesicular stomatitis virus, vaccinia virus, cowpox virus, Sindbis virus, Pixuna virus, Usutu virus, Inkoo virus, Puumalavirus, and Borna disease virus (BDV). The cell line was susceptible for all tested viruses, and most interestingly also for the difficult to propagate BDV.</p> <p>Conclusion</p> <p>In conclusion, the newly established cell line from wildlife rodents seems to be an excellent tool for the isolation and characterization of new rodent-associated viruses and may be used as <it>in vitro-</it>model to study properties and pathogenesis of these agents.</p

Motility of human renal cells is disturbed by infection with pathogenic hantaviruses

Background: Hemorrhagic fever with renal syndrome (HFRS) caused by pathogenic hantaviruses in Europe and Asia is often characterized by acute kidney injury (AKI) with massive proteinuria. Renal filtration depends on the integrity of epithelial and endothelial monolayers in the tubular and glomerular apparatus. Tubular and glomerular cells represent target cells of hantavirus infection. However, the detailed mechanisms of renal impairment induced by hantaviruses are not well understood. Methods: We analyzed the cellular consequences of hantavirus infection by measuring adhesion and migration capacity of human renal cells infected with Puumala (PUUV) or Hantaan (HTNV) virus. The impact of hantaviral nucleocapsid proteins (N proteins) on motility was examined by transfection of podocytes. Results: Infection of kidney cells with hantavirus species PUUV and HTNV causes a significant reduction of migration capacity. The impaired motility depends on viral replication and transfection of podocytes with N protein of PUUV or HTNV reveals that the expression of N protein alone is sufficient to deteriorate podocyte function. The cellular effects are more pronounced for the more pathogenic HTNV than for PUUV that causes a milder form of HFRS. Conclusions: The direct impairment of migration capacity of renal cells by hantaviral N proteins may contribute substantially to proteinuria observed in the clinical picture of hantavirus infection

Clinical characterization of two severe cases of hemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses Puumala and Dobrava-Belgrade genotype Sochi

Background: Hantavirus disease belongs to the emerging infections. The clinical picture and severity of infections differ between hantavirus species and may even vary between hantavirus genotypes. The mechanisms that lead to the broad variance of severity in infected patients are not completely understood. Host- and virus-specific factors are considered. Case presentation: We analyzed severe cases of hantavirus disease in two young women. The first case was caused by Puumala virus (PUUV) infection in Germany; the second case describes the infection with Dobrava-Belgrade virus (DOBV) in Russia. Symptoms, laboratory parameters and cytokine levels were analyzed and compared between the two patients. Serological and sequence analysis revealed that PUUV was the infecting agent for the German patient and the infection of the Russian patient was caused by Dobrava-Belgrade virus genotype Sochi (DOBV-Sochi). The symptoms in the initial phase of the diseases did not differ noticeably between both patients. However, deterioration of laboratory parameter values was prolonged and stronger in DOBV-Sochi than in PUUV infection. Circulating endothelial progenitor cells (cEPCs), known to be responsible for endothelial repair, were mobilized in both infections. Striking differences were observed in the temporal course and level of cytokine upregulation. Levels of angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), and stromal derived factor-1 (SDF-1α) were increased in both infections; but, sustained and more pronounced elevation was observed in DOBV-Sochi infection. Conclusions: Severe hantavirus disease caused by different hantavirus species did not differ in the general symptoms and clinical characteristics. However, we observed a prolonged clinical course and a late and enhanced mobilization of cytokines in DOBV-Sochi infection. The differences in cytokine deregulation may contribute to the observed variation in the clinical course

Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

BACKGROUND: The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. RESULTS: Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. CONCLUSION: Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo

Polar release of pathogenic Old World hantaviruses from renal tubular epithelial cells

Abstract Background Epithelio- and endotheliotropic viruses often exert polarized entry and release that may be responsible for viral spread and dissemination. Hantaviruses, mostly rodent-borne members of the Bunyaviridae family infect epithelial and endothelial cells of different organs leading to organ dysfunction or even failure. Endothelial and renal epithelial cells belong to the target cells of Old World hantavirus. Therefore, we examined the release of hantaviruses in several renal epithelial cell culture models. We used Vero cells that are commonly used in hantavirus studies and primary human renal epithelial cells (HREpC). In addition, we analyzed MDCKII cells, an epithelial cell line of a dog kidney, which represents a widely accepted in vitro model of polarized monolayers for their permissiveness for hantavirus infection. Results Vero C1008 and primary HREpCs were grown on porous-support filter inserts for polarization. Monolayers were infected with hantavirus Hantaan (HTNV) and Puumala (PUUV) virus. Supernatants from the apical and basolateral chamber of infected cells were analyzed for the presence of infectious particles by re-infection of Vero cells. Viral antigen and infectious particles of HTNV and PUUV were exclusively detected in supernatants collected from the apical chamber of infected Vero C1008 cells and HREpCs. MDCKII cells were permissive for hantavirus infection and polarized MDCKII cells released infectious hantaviral particles from the apical surface corresponding to the results of Vero and primary human epithelial cells. Conclusions Pathogenic Old World hantaviruses are released from the apical surface of different polarized renal epithelial cells. We characterized MDCKII cells as a suitable polarized cell culture model for hantavirus infection studies.</p

In-cell Western assay to quantify infection with pathogenic orthohantavirus Puumala virus in replication kinetics and antiviral drug testing

Hemorrhagic fever with renal syndrome (HFRS) represents a serious zoonotic disease caused by orthohantaviruses in Eurasia. A specific antiviral therapy is not available. HFRS is characterized by acute kidney injury (AKI) with often massive proteinuria. Infection of kidney cells may contribute to the clinical picture. However, orthohantaviral replication in kidney cells is not well characterized. Therefore, we aimed to perform a reliable high-throughput assay that allows the quantification of infection rates and testing of antiviral compounds in different cell types. We quantified relative infection rates of Eurasian pathogenic Puumala virus (PUUV) by staining of nucleocapsid protein (N protein) in an in-cell Western (ICW) assay. Vero E6 cells, derived from the African green monkey and commonly used in viral cell culture studies, and the human podocyte cell line CIHP (conditionally immortalized human podocytes) were used to test the ICW assay for replication kinetics and antiviral drug testing. Quantification of infection by ICW revealed reliable results for both cell types, as shown by their correlation with immunofluorescence quantification results by counting infected cells. Evaluation of antiviral efficacy of ribavirin by ICW assay revealed differences in the toxicity (TC) and inhibitory concentrations (IC) between Vero E6 cells and podocytes. IC5O of ribavirin in podocytes is about 12-fold lower than in Vero E6 cells. In summary, ICW assay together with relevant human target cells represents an important tool for the study of hantaviral replication and drug testing

Cells of the human respiratory tract support the replication of pathogenic Old World orthohantavirus Puumala

BACKGROUND: Transmission of all known pathogenic orthohantaviruses (family Hantaviridae) usually occurs via inhalation of aerosols contaminated with viral particles derived from infected rodents and organ manifestation of infections is characterized by lung and kidney involvement. Orthohantaviruses found in Eurasia cause hemorrhagic fever with renal syndrome (HFRS) and New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS). However, cases of infection with Old World orthohantaviruses with severe pulmonary manifestations have also been observed. Therefore, human airway cells may represent initial targets for orthohantavirus infection and may also play a role in the pathogenesis of infections with Eurasian orthohantaviruses. METHODS: We analyzed the permissiveness of primary endothelial cells of the human pulmonary microvasculature and of primary human epithelial cells derived from bronchi, bronchioles and alveoli for Old World orthohantavirus Puumala virus (PUUV) in vitro. In addition, we examined the expression of orthohantaviral receptors in these cell types. To minimize donor-specific effects, cells from two different donors were tested for each cell type. RESULTS: Productive infection with PUUV was observed for endothelial cells of the microvasculature and for the three tested epithelial cell types derived from different sites of the respiratory tract. Interestingly, infection and particle release were also detected in bronchial and bronchiolar epithelial cells although expression of the orthohantaviral receptor integrin β₃ was not detectable in these cell types. In addition, replication kinetics and viral release demonstrate enormous donor-specific variations. CONCLUSIONS: The human respiratory epithelium is among the first targets of orthohantaviral infection and may contribute to virus replication, dissemination and pathogenesis of HFRS-causing orthohantaviruses. Differences in initial pulmonary infection due to donor-specific factors may play a role in the observed broad variance of severity and symptoms of orthohantavirus disease in patients. The absence of detectable levels of integrin αVβ₃ surface expression on bronchial and small airway epithelial cells indicates an alternate mode of orthohantaviral entry in these cells that is independent from integrin β₃

Human Immunodeficiency Virus Type 1 Nef Activates p21-Activated Kinase via Recruitment into Lipid Rafts

The Nef protein of human immunodeficiency virus type 1 is an important factor in AIDS pathogenesis. In addition to downregulating CD4 and major histocompatibility complex class I molecules from the cell surface, as well as increasing virion infectivity, Nef triggers activation of the T-cell receptor (TCR) cascade to facilitate virus spread. Signaling pathways that are induced by Nef have been identified; however, it is unclear how and in which subcellular compartment Nef triggers signaling. Nef recruits a multiprotein complex to activate the cellular Pak kinase that mediates downstream effector functions. Since a subpopulation of Nef is present in detergent-insoluble microdomains (lipid rafts) from where physiological TCR signaling is initiated, we tested whether lipid rafts are instrumental for Nef-mediated Pak activation. In flotation analysis, Nef-associated Pak activity exclusively fractionated with lipid rafts. Activation of Pak in the presence of Nef coincided with lipid raft recruitment of the kinase, which was otherwise excluded from detergent-insoluble microdomains. Experimental solubilization of lipid rafts interfered with the association of Pak activity with Nef. To analyze the importance of the raft localization for Nef function more rigorously, we generated a palmitoylated Nef (PalmNef). PalmNef was highly enriched in lipid rafts and associated with significantly higher levels of Pak activity than Nef. Notably, activation of Pak by its physiological activators, Cdc42 and Rac, also occurred in lipid rafts and required raft integrity. Together, these data suggest that Nef induces signal transduction via the recruitment of a signaling machinery including Pak into lipid rafts, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade

Expression Profile of Human Renal Mesangial Cells Is Altered by Infection with Pathogenic Puumala Orthohantavirus

Acute kidney injury (AKI) with proteinuria is a hallmark of infections with Eurasian orthohantaviruses. Different kidney cells are identified as target cells of hantaviruses. Mesangial cells may play a central role in the pathogenesis of AKI by regulation of inflammatory mediators and signaling cascades. Therefore, we examined the characteristics of hantavirus infection on human renal mesangial cells (HRMCs). Receptor expression and infection with pathogenic Puumala virus (PUUV) and low-pathogenic Tula virus (TULV) were explored. To analyze changes in protein expression in infected mesangial cells, we performed a proteome profiler assay analyzing 38 markers of kidney damage. We compared the proteome profile of in vitro-infected HRMCs with the profile detected in urine samples of 11 patients with acute hantavirus infection. We observed effective productive infection of HRMCs with pathogenic PUUV, but only poor abortive infection for low-pathogenic TULV. PUUV infection resulted in the deregulation of proteases, adhesion proteins, and cytokines associated with renal damage. The urinary proteome profile of hantavirus patients demonstrated also massive changes, which in part correspond to the alterations observed in the in vitro infection of HRMCs. The direct infection of mesangial cells may induce a local environment of signal mediators that contributes to AKI in hantavirus infection