The Experts below are selected from a list of 1521 Experts worldwide ranked by ideXlab platform
Heinz Feldmann - One of the best experts on this subject based on the ideXlab platform.
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Neutralizing Monoclonal Antibodies Against the Gn and the Gc of the Andes Virus Glycoprotein Spike Complex Protect From Virus Challenge in a Preclinical Hamster Model
mBio, 2020Co-Authors: James Duehr, Meagan Mcmahon, Brandi N. Williamson, Fatima Amanat, Alan Durbin, David W. Hawman, Danny Noack, Skyler Uhl, Gene S. Tan, Heinz FeldmannAbstract:ABSTRACT HantaViruses are the etiological agent of hemorrhagic fever with renal syndrome (HFRS) and hantaVirus cardiopulmonary syndrome (HCPS). The latter is associated with case fatality rates ranging from 30% to 50%. HCPS cases are rare, with approximately 300 recorded annually in the Americas. Recently, an HCPS outbreak of unprecedented size has been occurring in and around Epuyen, in the southwestern Argentinian state of Chubut. Since November of 2018, at least 29 cases have been laboratory confirmed, and human-to-human transmission is suspected. Despite posing a significant threat to public health, no treatment or vaccine is available for hantaviral disease. Here, we describe an effort to identify, characterize, and develop neutralizing and protective antibodies against the glycoprotein complex (Gn and Gc) of Andes Virus (ANDV), the causative agent of the Epuyen outbreak. Using murine hybridoma technology, we generated 19 distinct monoclonal antibodies (MAbs) against ANDV GnGc. When tested for neutralization against a recombinant vesicular stomatitis Virus expressing the Andes glycoprotein (GP) (VSV-ANDV), 12 MAbs showed potent neutralization and 8 showed activity in an antibody-dependent cellular cytotoxicity reporter assay. Escape mutant analysis revealed that neutralizing MAbs targeted both the Gn and the Gc. Four MAbs that bound different epitopes were selected for preclinical studies and were found to be 100% protective against lethality in a Syrian hamster model of ANDV infection. These data suggest the existence of a wide array of neutralizing antibody epitopes on hantaVirus GnGc with unique properties and mechanisms of action. IMPORTANCE Infections with New World hantaViruses are associated with high case fatality rates, and no specific vaccine or treatment options exist. Furthermore, the biology of the hantaviral GnGc complex, its antigenicity, and its fusion machinery are poorly understood. Protective monoclonal antibodies against GnGc have the potential to be developed into therapeutics against hantaviral disease and are also great tools to elucidate the biology of the glycoprotein complex.
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Experimental Andes Virus infection in deer mice: Characteristics of infection and clearance in a heterologous rodent host. PLoS One 2013
2016Co-Authors: Jessica R Spengler, Brian Hjelle, Heinz Feldmann, Elaine Haddock, Don Gardner, Joseph PrescottAbstract:New World hantaViruses can cause hantaVirus cardiopulmonary syndrome with high mortality in humans. Distinct Virus species are hosted by specific rodent reservoirs, which also serve as the vectors. Although regional spillover has been documented, it is unknown whether rodent reservoirs are competent for infection by hantaViruses that are geographically separated, and known to have related, but distinct rodent reservoir hosts. We show that Andes Virus (ANDV) of South America, carried by the long tailed pygmy rice rat (Oligoryzomys longicaudatus), infects and replicates in vitro and in vivo in the deer mouse (Peromyscus maniculatus), the reservoir host of Sin Nombre Virus (SNV), found in North America. In experimentally infected deer mice, viral RNA was detected in the blood, lung, heart and spleen, but Virus was cleared by 56 days post inoculation (dpi). All of the inoculated deer mice mounted a humoral immune response by 14 dpi, and produced measurable amounts of neutralizing antibodies by 21 dpi. An up-regulation of Ccl3, Ccl4, Ccl5, and Tgfb, a strong CD4+ T-cell response, and down-regulation of Il17, Il21 and Il23 occurred during infection. Infection was transient with an absence of clinical signs or histopathological changes. This is the first evidence that ANDV asymptomatically infects, and is immunogenic in deer mice, a non-natural host species of ANDV. Comparing the immune response in this model to that of the immune response in the natural hosts upon infection with their co-adapted hantaViruses may help clarify th
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Long-term single-dose efficacy of a vesicular stomatitis Virus-based Andes Virus vaccine in Syrian hamsters.
Viruses, 2014Co-Authors: Joseph Prescott, Kyle S. Brown, Blair L. Debuysscher, Heinz FeldmannAbstract:Andes Virus (ANDV) is highly pathogenic in humans and is the primary etiologic agent of hantaVirus cardiopulmonary syndrome (HCPS) in South America. Case-fatality rates are as high as 50% and there are no approved vaccines or specific therapies for infection. Our laboratory has recently developed a replication-competent recombinant vesicular stomatitis Virus (VSV)-based vaccine that expressed the glycoproteins of Andes Virus in place of the native VSV glycoprotein (G). This vaccine is highly efficacious in the Syrian hamster model of HCPS when given 28 days before challenge with ANDV, or when given around the time of challenge (peri-exposure), and even protects when administered post-exposure. Herein, we sought to test the durability of the immune response to a single dose of this vaccine in Syrian hamsters. This vaccine was efficacious in hamsters challenged intranasally with ANDV 6 months after vaccination (p = 0.025), but animals were not significantly protected following 1 year of vaccination (p = 0.090). The decrease in protection correlated with a reduction of measurable neutralizing antibody responses, and suggests that a more robust vaccination schedule might be required to provide long-term immunity.
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Expression profiling of lymph node cells from deer mice infected with Andes Virus
BMC Immunology, 2013Co-Authors: Tony Schountz, Heinz Feldmann, Timothy I Shaw, Travis C Glenn, Joseph PrescottAbstract:Background Deer mice ( Peromyscus maniculatus ) are the principal reservoir hosts of Sin Nombre Virus (SNV), the cause of the great majority of hantaVirus cardiopulmonary syndrome (HCPS) cases in North America. SNV, like all hantaViruses with their reservoirs, causes persistent infection without pathology in deer mice and appear to elicit a regulatory T cell response. Deer mice are also susceptible to Andes Virus (ANDV), which causes the great majority of HCPS cases in South America, but they clear infection by 56 days post infection without signs of disease. Results We examined lymph node cell responses of deer mice infected with ANDV to determine expression profiles upon in vitro recall challenge with viral antigen. Because the deer mouse genome is currently unannotated, we developed a bioinformatics pipeline to use known lab mouse ( Mus musculus ) cDNAs to predict genes within the deer mouse genome and design primers for quantitative PCR ( http://dna.publichealth.uga.edu/BlastPrimer/BlastPrimer.php ). Of 94 genes examined, 20 were elevated, the plurality of which were Th2-specific, whereas 12 were downregulated. Other expressed genes represented Th1, regulatory T cells and follicular helper T cells, and B cells, but not Th17 cells, indicating that many cellular phenotypes participate in the host response to Andes Virus. Conclusions The ability to examine expression levels of nearly any gene from deer mice should allow direct comparison of infection with SNV or ANDV to determine the immunological pathways used for clearance of hantaVirus infection in a reservoir host species.
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Expression profiling of lymph node cells from deer mice infected with Andes Virus
BMC immunology, 2013Co-Authors: Tony Schountz, Heinz Feldmann, Timothy I Shaw, Travis C Glenn, Joseph PrescottAbstract:Deer mice (Peromyscus maniculatus) are the principal reservoir hosts of Sin Nombre Virus (SNV), the cause of the great majority of hantaVirus cardiopulmonary syndrome (HCPS) cases in North America. SNV, like all hantaViruses with their reservoirs, causes persistent infection without pathology in deer mice and appear to elicit a regulatory T cell response. Deer mice are also susceptible to Andes Virus (ANDV), which causes the great majority of HCPS cases in South America, but they clear infection by 56 days post infection without signs of disease. We examined lymph node cell responses of deer mice infected with ANDV to determine expression profiles upon in vitro recall challenge with viral antigen. Because the deer mouse genome is currently unannotated, we developed a bioinformatics pipeline to use known lab mouse (Mus musculus) cDNAs to predict genes within the deer mouse genome and design primers for quantitative PCR ( http://dna.publichealth.uga.edu/BlastPrimer/BlastPrimer.php ). Of 94 genes examined, 20 were elevated, the plurality of which were Th2-specific, whereas 12 were downregulated. Other expressed genes represented Th1, regulatory T cells and follicular helper T cells, and B cells, but not Th17 cells, indicating that many cellular phenotypes participate in the host response to Andes Virus. The ability to examine expression levels of nearly any gene from deer mice should allow direct comparison of infection with SNV or ANDV to determine the immunological pathways used for clearance of hantaVirus infection in a reservoir host species.
Jay W. Hooper - One of the best experts on this subject based on the ideXlab platform.
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Innate immune responses elicited by Sin Nombre Virus or type I IFN agonists protect hamsters from lethal Andes Virus infections.
The Journal of general virology, 2018Co-Authors: Rebecca L. Brocato, Anita K. Mcelroy, Jeffrey M. Smith, Victoria Wahl, Christopher D. Hammerbeck, Matthew Josleyn, Jay W. HooperAbstract:Sin Nombre Virus (SNV) and Andes Virus (ANDV) cause hantaVirus pulmonary syndrome (HPS) in humans. Both SNV and ANDV infect Syrian hamsters, but only ANDV causes lethal disease. A co-infection study was performed to determine which Virus, SNV or ANDV, would dominate the survival outcome in hamsters. Infection of hamsters with SNV 1 day before ANDV challenge did not result in disease characteristic of the latter Virus, and all animals survived challenge. Control animals infected solely with ANDV all succumbed by day 14. In contrast, when Viruses were injected at the same site concurrently, all hamsters succumbed to HPS disease. HantaViruses are segmented Viruses; therefore we investigated which segment might be responsible for the protective phenotype of SNV by using two SNV/ANDV reassortant Viruses, both with reciprocal M-segments from the other Virus (denoted ASA and SAS). Both reassortants asymptomatically infect hamsters, similar to SNV. However, unlike SNV, 1 day prior preinfection with the reassortant Virus did not prevent ANDV lethality. The ASA reassortant Virus, but not SAS, protected hamsters from lethal ANDV infection when administered 3 days prior to ANDV challenge. Similar to SNV preinfection, the potent innate immune stimulator poly I:C administered to hamsters 1 day before ANDV challenge prevented lethal ANDV disease. Combined, these results suggest that the difference in pathogenicity of SNV and ANDV in hamsters involves differences in early host-pathogen interactions and resultant anti-viral immune responses of both the innate and adaptive immune system.
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A lethal disease model for New World hantaViruses using immunosuppressed Syrian hamsters.
PLoS neglected tropical diseases, 2017Co-Authors: Valentijn Vergote, Jay W. Hooper, Lies Laenen, Bert Vanmechelen, Marc Van Ranst, Erik Verbeken, Piet MaesAbstract:Background HantaVirus, the hemorrhagic causative agent of two clinical diseases, is found worldwide with variation in severity, incidence and mortality. The most lethal hantaViruses are found on the American continent where the most prevalent Viruses like Andes Virus and Sin Nombre Virus are known to cause hantaVirus pulmonary syndrome. New World hantaVirus infection of immunocompetent hamsters results in an asymptomatic infection except for Andes Virus and Maporal Virus; the only hantaViruses causing a lethal disease in immunocompetent Syrian hamsters mimicking hantaVirus pulmonary syndrome in humans. Methodology/Principal findings Hamsters, immunosuppressed with dexamethasone and cyclophosphamide, were infected intramuscularly with different New World hantaVirus strains (Bayou Virus, Black Creek Canal Virus, Cano Delgadito Virus, Choclo Virus, Laguna Negra Virus, and Maporal Virus). In the present study, we show that immunosuppression of hamsters followed by infection with a New World hantaVirus results in an acute disease that precisely mimics both hantaVirus disease in humans and Andes Virus infection of hamsters. Conclusions/ Significance Infected hamsters showed specific clinical signs of disease and moreover, histological analysis of lung tissue showed signs of pulmonary edema and inflammation within alveolar septa. In this study, we were able to infect immunosuppressed hamsters with different New World hantaViruses reaching a lethal outcome with signs of disease mimicking human disease.
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Gastrointestinal Tract As Entry Route for HantaVirus Infection
Frontiers in microbiology, 2017Co-Authors: Peter T. Witkowski, Jay W. Hooper, Rebecca L. Brocato, Casey C. Perley, Christian Jürgensen, Jörg-dieter Schulzke, Detlev H. Krüger, Roland BückerAbstract:Background. HantaViruses are zoonotic agents that cause hemorrhagic fevers and are thought to be transmitted to humans by exposure to aerosolized excreta of infected rodents. Puumala Virus (PUUV) is the predominant endemic hantaVirus in Europe. A large proportion of PUUV-infected patients suffer from gastrointestinal symptoms of unclear origin. In this study we demonstrate that PUUV infection can occur via the alimentary tract. Methods. We investigated susceptibility of the human small intestinal epithelium for PUUV infection and analyzed the resistance of virions to gastric juice. As model for intestinal Virus translocation we performed infection experiments with human intestinal Caco-2 monolayers. In animal experiments we infected Syrian hamsters with PUUV via the intragastric route and tested seroconversion and protective immunity against subsequent Andes Virus challenge. Results. PUUV retained infectivity in gastric juice at pH >3. The Virus invaded Caco-2 monolayers in association with endosomal antigen EEA1, followed by Virus replication and loss of epithelial barrier function with basolateral Virus occurrence. Cellular disturbance and depletion of the tight junction protein ZO-1 appeared after prolonged infection, leading to paracellular leakage (leak flux diarrhea). Moreover, animal experiments led to dose-dependent seroconversion and protection against lethal Andes Virus challenge. Conclusions. We provide evidence that hantaVirus can infect the organism via the alimentary tract and suggest a novel aspect of hantaVirus infection and pathogenesis. Significance. HantaViruses are zoonotic pathogens causing severe hemorrhagic fevers worldwide. They are transmitted to humans by small mammals. To date these Viruses were thought to infect exclusively through the airborne route by inhalation of aerosols from infectious animal droppings or by rodent bites. In our work we could show that the alimentary tract is an alternative path of infection for hantaViruses, meaning a new association of Virus and disease. These findings have impact on current textbook knowledge and bring many implications for hantaVirus epidemiology and outbreak prevention measures.
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A lethal disease model for New World hantaViruses using immunosuppressed Syrian hamsters.
Public Library of Science (PLoS), 2017Co-Authors: Valentijn Vergote, Jay W. Hooper, Lies Laenen, Bert Vanmechelen, Marc Van Ranst, Erik Verbeken, Piet MaesAbstract:HantaVirus, the hemorrhagic causative agent of two clinical diseases, is found worldwide with variation in severity, incidence and mortality. The most lethal hantaViruses are found on the American continent where the most prevalent Viruses like Andes Virus and Sin Nombre Virus are known to cause hantaVirus pulmonary syndrome. New World hantaVirus infection of immunocompetent hamsters results in an asymptomatic infection except for Andes Virus and Maporal Virus; the only hantaViruses causing a lethal disease in immunocompetent Syrian hamsters mimicking hantaVirus pulmonary syndrome in humans.Hamsters, immunosuppressed with dexamethasone and cyclophosphamide, were infected intramuscularly with different New World hantaVirus strains (Bayou Virus, Black Creek Canal Virus, Caño Delgadito Virus, Choclo Virus, Laguna Negra Virus, and Maporal Virus). In the present study, we show that immunosuppression of hamsters followed by infection with a New World hantaVirus results in an acute disease that precisely mimics both hantaVirus disease in humans and Andes Virus infection of hamsters.Infected hamsters showed specific clinical signs of disease and moreover, histological analysis of lung tissue showed signs of pulmonary edema and inflammation within alveolar septa. In this study, we were able to infect immunosuppressed hamsters with different New World hantaViruses reaching a lethal outcome with signs of disease mimicking human disease
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ribavirin protects syrian hamsters against lethal hantaVirus pulmonary syndrome after intranasal exposure to Andes Virus
Viruses, 2013Co-Authors: Monica M. Ogg, Colleen B. Jonsson, Jeremy V. Camp, Jay W. HooperAbstract:Andes Virus, ANDV, harbored by wild rodents, causes the highly lethal hantaVirus pulmonary syndrome (HPS) upon transmission to humans resulting in death in 30% to 50% of the cases. As there is no treatment for this disease, we systematically tested the efficacy of ribavirin in vitro and in an animal model. In vitro assays confirmed antiviral activity and determined that the most effective doses were 40 µg/mL and above. We tested three different concentrations of ribavirin for their capability to prevent HPS in the ANDV hamster model following an intranasal challenge. While the highest level of ribavirin (200 mg/kg) was toxic to the hamster, both the middle (100 mg/kg) and the lowest concentration (50 mg/kg) prevented HPS in hamsters without toxicity. Specifically, 8 of 8 hamsters survived intranasal challenge for both of those groups whereas 7 of 8 PBS control-treated animals developed lethal HPS. Further, we report that administration of ribavirin at 50 mg/kg/day starting on days 6, 8, 10, or 12 post-infection resulted in significant protection against HPS in all groups. Administration of ribavirin at 14 days post-infection also provided a significant level of protection against lethal HPS. These data provide in vivo evidence supporting the potential use of ribavirin as a post-exposure treatment to prevent HPS after exposure by the respiratory route.
David Safronetz - One of the best experts on this subject based on the ideXlab platform.
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Vesicular Stomatitis Virus-Based Vaccines Provide Cross-Protection against Andes and Sin Nombre Viruses
Viruses, 2019Co-Authors: Bryce M. Warner, Derek R. Stein, Rohit K. Jangra, Megan M. Slough, Patrycja Sroga, Angela Sloan, Kathy L Frost, Stephanie A. Booth, Kartik Chandran, David SafronetzAbstract:Andes Virus (ANDV) and Sin Nombre Virus (SNV) are the main causative agents responsible for hantaVirus cardiopulmonary syndrome (HCPS) in the Americas. HCPS is a severe respiratory disease with a high fatality rate for which there are no approved therapeutics or vaccines available. Some vaccine approaches for HCPS have been tested in preclinical models, but none have been tested in infectious models in regard to their ability to protect against multiple species of HCPS-causing Viruses. Here, we utilize recombinant vesicular stomatitis Virus-based (VSV) vaccines for Andes Virus (ANDV) and Sin Nombre Virus (SNV) and assess their ability to provide cross-protection in infectious challenge models. We show that, while both rVSVΔG/ANDVGPC and rVSVΔG/SNVGPC display attenuated growth as compared to wild type VSV, each vaccine is able to induce a cross-reactive antibody response. Both vaccines protected against both homologous and heterologous challenge with ANDV and SNV and prevented HCPS in a lethal ANDV challenge model. This study provides evidence that the development of a single vaccine against HCPS-causing hantaViruses could provide protection against multiple agents.
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Hamster-Adapted Sin Nombre Virus Causes Disseminated Infection and Efficiently Replicates in Pulmonary Endothelial Cells without Signs of Disease
Journal of virology, 2013Co-Authors: David Safronetz, Heinz Feldmann, Joseph Prescott, Elaine Haddock, Dana P. Scott, Hideki EbiharaAbstract:To date, a laboratory animal model for the study of Sin Nombre Virus (SNV) infection or associated disease has not been described. Unlike infection with Andes Virus, which causes lethal hantaVirus pulmonary syndrome (HPS)-like disease in hamsters, SNV infection is short-lived, with no viremia and little dissemination. Here we investigated the effect of passaging SNV in hamsters. We found that a host-adapted SNV achieves prolonged and disseminated infection in hamsters, including efficient replication in pulmonary endothelial cells, albeit without signs of disease.
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The Syrian hamster model of hantaVirus pulmonary syndrome.
Antiviral research, 2012Co-Authors: David Safronetz, Heinz Feldmann, Hideki Ebihara, Jay W. HooperAbstract:HantaVirus pulmonary syndrome (HPS) is a relatively rare, but frequently fatal disease associated with New World hantaViruses, most commonly Sin Nombre and Andes Viruses in North and South America, respectively. It is characterized by fever and the sudden, rapid onset of severe respiratory distress and cardiogenic shock, which can be fatal in up to 50% of cases. Currently there are no approved antiviral therapies or vaccines for the treatment or prevention of HPS. A major obstacle in the development of effective medical countermeasures against highly pathogenic agents like the hantaViruses is recapitulating the human disease as closely as possible in an appropriate and reliable animal model. To date, the only animal model that resembles HPS in humans is the Syrian hamster model. Following infection with Andes Virus, hamsters develop HPS-like disease which faithfully mimics the human condition with respect to incubation period and pathophysiology of disease. Perhaps most importantly, the sudden and rapid onset of severe respiratory distress observed in humans also occurs in hamsters. The last several years has seen an increase in studies utilizing the Andes Virus hamster model which have provided unique insight into HPS pathogenesis as well as potential therapeutic and vaccine strategies to treat and prevent HPS. The purpose of this article is to review the current understanding of HPS disease progression in Syrian hamsters and discuss the suitability of utilizing this model to evaluate potential medical countermeasures against HPS.
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Pathogenesis and Host Response in Syrian Hamsters following Intranasal Infection with Andes Virus
PLoS pathogens, 2011Co-Authors: David Safronetz, Marko Zivcec, Rachel Lacasse, Friederike Feldmann, Rebecca Rosenke, Dan Long, Elaine Haddock, Douglas Brining, Donald J. Gardner, Heinz FeldmannAbstract:HantaVirus pulmonary syndrome (HPS), also referred to as hantaVirus cardiopulmonary syndrome (HCPS), is a rare but frequently fatal disease caused by New World hantaViruses. In humans HPS is associated with severe pulmonary edema and cardiogenic shock; however, the pathogenesis of this disease remains unclear largely due to a lack of suitable animal models for the study of disease progression. In this study we monitored clinical, virological, pathophysiological parameters and host immunological responses to decipher pathological factors and events in the lethal Syrian hamster model of HPS following intranasal inoculation of Andes Virus. Transcriptional profiling of the host gene responses demonstrated a suppression of innate immune responses in most organs analyzed during the early stage of infection, except for in the lung which had low level activation of several pro-inflammatory genes. During this phase Andes Virus established a systemic infection in hamsters, with viral antigen readily detectable in the endothelium of the majority of tissues analyzed by 7–8 days post-inoculation. Despite wide-spread infection, histological analysis confirmed pathological abnormalities were almost exclusively found in the lungs. Immediately preceding clinical signs of disease, intense activation of pro-inflammatory and Th1/Th2 responses were observed in the lungs as well as the heart, but not in peripheral organs, suggesting that localized immune-modulations by infection is paramount to pathogenesis. Throughout the course of infection a strong suppression of regulatory T-cell responses was noted and is hypothesized to be the basis of the aberrant immune activations. The unique and comprehensive monitoring of host immune responses to hantaVirus infection increases our understanding of the immuno-pathogenesis of HPS and will facilitate the development of treatment strategies targeting deleterious host immunological responses.
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vesicular stomatitis Virus based vaccine protects hamsters against lethal challenge with Andes Virus
Journal of Virology, 2011Co-Authors: Kyle S. Brown, Hideki Ebihara, Heinz Feldmann, David Safronetz, Andrea MarziAbstract:Andes Virus (ANDV) is a highly pathogenic South American hantaVirus that causes hantaVirus pulmonary syndrome (HPS). A high case fatality rate, the potential for human-to-human transmission, the capacity to infect via aerosolization, and the absence of effective therapies make it imperative that a safe, fast-acting, and effective ANDV vaccine be developed. We generated and characterized a recombinant vesicular stomatitis Virus (VSV) vector expressing the ANDV surface glycoprotein precursor (VSVΔG/ANDVGPC) as a possible vaccine candidate and tested its efficacy in the only lethal-disease animal model of HPS. Syrian hamsters immunized with a single injection of VSVΔG/ANDVGPC were fully protected against disease when challenged at 28, 14, 7, or 3 days postimmunization with a lethal dose of ANDV; however, the mechanism of protection seems to differ depending on when the immunization occurs. At 28 days postimmunization, a lack of detectable ANDV RNA in lung, liver, and blood tissue samples, as well as a lack of seroconversion to the ANDV nucleocapsidprotein in nearly all animals, suggested largely sterile immunity. The vaccine was able to generate high levels of neutralizing anti-ANDV GN/GC antibodies, which seem to play a role as a mechanism of vaccine protection. Administration of the vaccine at 7 or 3 days before challenge also resulted in full protection but with no specific neutralizing humoral immune response, suggesting a possible role of innate responses in protection against challenge Virus replication. Administration of the vaccine 24 h postchallenge was successful in protecting 90% of hamsters and again suggested the induction of a potent antiviral state by the recombinant vector as a potential mechanism. Overall, our data suggest the potential for the use of the VSV platform as a fast-acting and effective prophylaxis/postexposure treatment against lethal hantaVirus infections.
Erich R Mackow - One of the best experts on this subject based on the ideXlab platform.
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Unique Interferon Pathway Regulation by the Andes Virus Nucleocapsid Protein Is Conferred by Phosphorylation of Serine 386.
Journal of virology, 2019Co-Authors: Matthew J. Simons, Elena E Gorbunova, Erich R MackowAbstract:Andes Virus (ANDV) causes hantaVirus pulmonary syndrome (HPS) and is the only hantaVirus shown to spread person to person and cause a highly lethal HPS-like disease in Syrian hamsters. The unique ability of ANDV N protein to inhibit beta interferon (IFNβ) induction may contribute to its virulence and spread. Here we analyzed IFNβ regulation by ANDV N protein substituted with divergent residues from the nearly identical Maporal Virus (MAPV) N protein. We found that MAPV N fails to inhibit IFNβ signaling and that replacing ANDV residues 252 to 296 with a hypervariable domain (HVD) from MAPV N prevents IFNβ regulation. In addition, changing ANDV residue S386 to the histidine present in MAPV N or the alanine present in other hantaViruses prevented ANDV N from regulating IFNβ induction. In contrast, replacing serine with phosphoserine-mimetic aspartic acid (S386D) in ANDV N robustly inhibited interferon regulatory factor 3 (IRF3) phosphorylation and IFNβ induction. Additionally, the MAPV N protein gained the ability to inhibit IRF3 phosphorylation and IFNβ induction when ANDV HVD and H386D replaced MAPV residues. Mass spectroscopy analysis of N protein from ANDV-infected cells revealed that S386 is phosphorylated, newly classifying ANDV N as a phosphoprotein and phosphorylated S386 as a unique determinant of IFN regulation. In this context, the finding that the ANDV HVD is required for IFN regulation by S386 but dispensable for IFN regulation by D386 suggests a role for HVD in kinase recruitment and S386 phosphorylation. These findings delineate elements within the ANDV N protein that can be targeted to attenuate ANDV and suggest targeting cellular kinases as potential ANDV therapeutics. IMPORTANCE ANDV contains virulence determinants that uniquely permit it to spread person to person and cause highly lethal HPS in immunocompetent hamsters. We discovered that ANDV S386 and an ANDV-specific hypervariable domain permit ANDV N to inhibit IFN induction and that IFN regulation is directed by phosphomimetic S386D substitutions in ANDV N. In addition, MAPV N proteins containing D386 and ANDV HVD gained the ability to inhibit IFN induction. Validating these findings, mass spectroscopy analysis revealed that S386 of ANDV N protein is uniquely phosphorylated during ANDV infection. Collectively, these findings reveal new paradigms for ANDV N protein as a phosphoprotein and IFN pathway regulator and suggest new mechanisms for hantaVirus regulation of cellular kinases and signaling pathways. Our findings define novel IFN-regulating virulence determinants of ANDV, identify residues that can be modified to attenuate ANDV for vaccine development, and suggest the potential for kinase inhibitors to therapeutically restrict ANDV replication.
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The Andes Virus Nucleocapsid Protein Directs Basal Endothelial Cell Permeability by Activating RhoA.
mBio, 2016Co-Authors: Elena E Gorbunova, Irina N Gavrilovskaya, Matthew J. Simons, Erich R MackowAbstract:ABSTRACT Andes Virus (ANDV) predominantly infects microvascular endothelial cells (MECs) and nonlytically causes an acute pulmonary edema termed hantaVirus pulmonary syndrome (HPS). In HPS patients, virtually every pulmonary MEC is infected, MECs are enlarged, and infection results in vascular leakage and highly lethal pulmonary edema. We observed that MECs infected with the ANDV hantaVirus or expressing the ANDV nucleocapsid (N) protein showed increased size and permeability by activating the Rheb and RhoA GTPases. Expression of ANDV N in MECs increased cell size by preventing tuberous sclerosis complex (TSC) repression of Rheb-mTOR-pS6K. N selectively bound the TSC2 N terminus (1 to 1403) within a complex containing TSC2/TSC1/TBC1D7, and endogenous TSC2 reciprocally coprecipitated N protein from ANDV-infected MECs. TSCs normally restrict RhoA-induced MEC permeability, and we found that ANDV infection or N protein expression constitutively activated RhoA. This suggests that the ANDV N protein alone is sufficient to activate signaling pathways that control MEC size and permeability. Further, RhoA small interfering RNA, dominant-negative RhoA(N19), and the RhoA/Rho kinase inhibitors fasudil and Y27632 dramatically reduced the permeability of ANDV-infected MECs by 80 to 90%. Fasudil also reduced the bradykinin-directed permeability of ANDV and Hantaan Virus-infected MECs to control levels. These findings demonstrate that ANDV activation of RhoA causes MEC permeability and reveal a potential edemagenic mechanism for ANDV to constitutively inhibit the basal barrier integrity of infected MECs. The central importance of RhoA activation in MEC permeability further suggests therapeutically targeting RhoA, TSCs, and Rac1 as potential means of resolving capillary leakage during hantaVirus infections. IMPORTANCE HPS is hallmarked by acute pulmonary edema, hypoxia, respiratory distress, and the ubiquitous infection of pulmonary MECs that occurs without disrupting the endothelium. Mechanisms of MEC permeability and targets for resolving lethal pulmonary edema during HPS remain enigmatic. Our findings suggest a novel underlying mechanism of MEC dysfunction resulting from ANDV activation of the Rheb and RhoA GTPases that, respectively, control MEC size and permeability. Our studies show that inhibition of RhoA blocks ANDV-directed permeability and implicate RhoA as a potential therapeutic target for restoring capillary barrier function to the ANDV-infected endothelium. Since RhoA activation forms a downstream nexus for factors that cause capillary leakage, blocking RhoA activation is liable to restore basal capillary integrity and prevent edema amplified by tissue hypoxia and respiratory distress. Targeting the endothelium has the potential to resolve disease during symptomatic stages, when replication inhibitors lack efficacy, and to be broadly applicable to other hemorrhagic and edematous viral diseases.
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An innate immunity-regulating virulence determinant is uniquely encoded by the Andes Virus nucleocapsid protein.
mBio, 2014Co-Authors: Velasco Cimica, Nadine A. Dalrymple, Eric Roth, Aleksandr Nasonov, Erich R MackowAbstract:ABSTRACT Andes Virus (ANDV) is the only hantaVirus known to spread from person to person and shown to cause highly lethal hantaVirus pulmonary syndrome (HPS) in patients and Syrian hamsters. HantaViruses replicate in human endothelial cells and accomplish this by restricting the early induction of beta interferon (IFN-β)- and IFN-stimulated genes (ISGs). Our studies reveal that the ANDV nucleocapsid (N) protein uniquely inhibits IFN signaling responses directed by cytoplasmic double-stranded RNA (dsRNA) sensors RIG-I and MDA5. In contrast, N proteins from Sin Nombre, New York-1, and Prospect Hill hantaViruses had no effect on RIG-I/MDA5-directed transcriptional responses from IFN-β-, IFN-stimulated response element (ISRE)-, or κB-containing promoters. Ablating a potential S-segment nonstructural open reading frame (ORF) (NSs) within the ANDV plasmid expressing N protein failed to alter IFN regulation by ANDV N protein. Further analysis demonstrated that expressing the ANDV N protein inhibited downstream IFN pathway activation directed by MAVS, TBK1, and IκB kinase e (IKKe) but failed to inhibit transcriptional responses directed by constitutive expression of active interferon regulatory factor IRF3-5D or after stimulation by alpha interferon (IFN-α) or tumor necrosis factor alpha (TNF-α). Consistent with IFN pathway-specific regulation, the ANDV N protein inhibited TBK1-directed IRF3 phosphorylation (phosphorylation of serine 396 [pS396]) and TBK1 autophosphorylation (pS172). Collectively, these findings indicate that the ANDV N inhibits IFN signaling responses by interfering with TBK1 activation, upstream of IRF3 phosphorylation and NF-κB activation. Moreover, our findings reveal that ANDV uniquely carries a gene encoding a virulence determinant within its N protein that is capable of restricting ISG and IFN-β induction and provide a rationale for the novel pathogenesis and spread of ANDV. IMPORTANCE Andes Virus (ANDV) is distinguished from other hantaViruses by its unique ability to spread from person to person and cause lethal hantaVirus pulmonary syndrome (HPS)-like disease in Syrian hamsters. However, virulence determinants that distinguish ANDV from other pathogenic hantaViruses have yet to be defined. Here we reveal that ANDV uniquely contains a virulence determinant within its nucleocapsid (N) protein that potently inhibits innate cellular signaling pathways. This novel function of the N protein provides a new mechanism for hantaViruses to regulate interferon (IFN) and IFN-stimulated gene (ISG) induction that is likely to contribute to the enhanced ability of ANDV to replicate, spread, and cause disease. These findings differentiate ANDV from other HPS-causing hantaViruses and provide a potential target for viral attenuation that needs to be considered in vaccine development.
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Hypoxia Induces Permeability and Giant Cell Responses of Andes Virus-Infected Pulmonary Endothelial Cells by Activating the mTOR-S6K Signaling Pathway
Journal of virology, 2013Co-Authors: Irina N Gavrilovskaya, Elena E Gorbunova, Erich R MackowAbstract:Andes Virus (ANDV) is a South American hantaVirus that causes a highly lethal hantaVirus pulmonary syndrome (HPS) characterized by hypoxia, thrombocytopenia, and vascular leakage leading to acute pulmonary edema. ANDV infects human pulmonary microvascular and lymphatic endothelial cells (MECs and LECs, respectively) and nonlytically enhances the permeability of interendothelial cell adherence junctions in response to vascular endothelial growth factor (VEGF). Recent findings also indicate that ANDV causes the formation of giant endothelial cells. Here, we demonstrate that hypoxic conditions alone enhance permeability and giant cell responses of ANDV-infected MECs and LECs through activation of the mTOR signaling pathway. In contrast to infection of cells with nonpathogenic Tula Virus (TULV), we observed that exposure of ANDV-infected MECs and LECs to hypoxic conditions resulted in a 3- to 6-fold increase in monolayer permeability and the formation of giant cells 3× to 5× normal size. ANDV infection in combination with hypoxic conditions resulted in the enhancement of hypoxia-inducible factor 1α (HIF1α)-directed VEGF A, angiopoietin 4, and EGLN3 transcriptional responses. Constitutive mTOR signaling induces the formation of giant cells via phosphorylation of S6K, and mTOR regulates hypoxia and VEGF A-induced cellular responses. We found that S6K was hyperphosphorylated in ANDV-infected, hypoxia-treated MECs and LECs and that rapamycin treatment for 1 h inhibited mTOR signaling responses and blocked permeability and giant cell formation in ANDV-infected monolayers. These findings indicate that ANDV infection and hypoxic conditions enhance mTOR signaling responses, resulting in enhanced endothelial cell permeability and suggest a role for rapamycin in therapeutically stabilizing the endothelium of microvascular and lymphatic vessels during ANDV infection.
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Andes Virus infection of lymphatic endothelial cells causes giant cell and enhanced permeability responses that are rapamycin and vascular endothelial growth factor C sensitive.
Journal of virology, 2012Co-Authors: Irina N Gavrilovskaya, Elena E Gorbunova, Erich R MackowAbstract:HantaViruses primarily infect endothelial cells (ECs) and nonlytically cause vascular changes that result in hemorrhagic fever with renal syndrome (HFRS) and hantaVirus pulmonary syndrome (HPS). Acute pulmonary edema during HPS may be caused by capillary leakage and failure of lymphatic vessels to clearfluids. Uniquely regulated lymphatic ECs (LECs) controlfluid clearance, although roles for lymphatics in hantaVirus disease remain undetermined. Here we report that hantaViruses productively infect LECs and that LEC infection by HPS causing Andes Virus (ANDV) and HFRS causing Hantaan Virus (HTNV) are inhibited by v3integrin antibodies. Although v3integrins regulate permeabilizing responses directed by vascular endothelial growth factor receptor 2 (VEGFR2), we found that only ANDV-infected LECs were hyperpermeabilized by the addition of VEGF-A. However, VEGF-C activation of LEC-specific VEGFR3 receptors blocked ANDV- and VEGF-A-induced LEC permeability. In addition,75% of ANDV-infected LECs became viable mononuclear giant cells, >4 times larger than normal, in response to VEGF-A. Giant cells are associated with constitutive mammalian target of rapamycin (mTOR) activation, and we found that both giant LECs and LEC permeability were sensitive to rapamycin, an mTOR inhibitor, and VEGF-C addition. Thesefindings indicate that ANDV uniquely alters VEGFR2-mTOR signaling responses of LECs, resulting in giant cell and LEC permeability responses. This suggests that ANDV infection alters normal LEC and lymphatic vessel functions which may contribute to edematousfluid accumulation during HPS. Moreover, the ability of VEGF-C and rapamycin to normalize LEC responses suggests a potential therapeutic approach for reducing pulmonary edema and the severity of HPS following ANDV infection.
Christina F. Spiropoulou - One of the best experts on this subject based on the ideXlab platform.
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Endocytic Pathways Used by Andes Virus to Enter Primary Human Lung Endothelial Cells.
PloS one, 2016Co-Authors: Cheng-feng Chiang, Mike Flint, Jin-mann S. Lin, Christina F. SpiropoulouAbstract:Andes Virus (ANDV) is the major cause of hantaVirus pulmonary syndrome (HPS) in South America. Despite a high fatality rate (up to 40%), no vaccines or antiviral therapies are approved to treat ANDV infection. To understand the role of endocytic pathways in ANDV infection, we used 3 complementary approaches to identify cellular factors required for ANDV entry into human lung microvascular endothelial cells. We screened an siRNA library targeting 140 genes involved in membrane trafficking, and identified 55 genes required for ANDV infection. These genes control the major endocytic pathways, endosomal transport, cell signaling, and cytoskeleton rearrangement. We then used infectious ANDV and retroviral pseudovirions to further characterize the possible involvement of 9 of these genes in the early steps of ANDV entry. In addition, we used markers of cellular endocytosis along with chemical inhibitors of known endocytic pathways to show that ANDV uses multiple routes of entry to infect target cells. These entry mechanisms are mainly clathrin-, dynamin-, and cholesterol-dependent, but can also occur via a clathrin-independent manner.
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Effect of Vandetanib on Andes Virus survival in the hamster model of HantaVirus pulmonary syndrome.
Antiviral Research, 2016Co-Authors: Brian H. Bird, Punya Shrivastava-ranjan, Kimberly A. Dodd, Bobbie R. Erickson, Christina F. SpiropoulouAbstract:Abstract HantaVirus pulmonary syndrome (HPS) is a severe disease caused by hantaVirus infection of pulmonary microvascular endothelial cells leading to microvascular leakage, pulmonary edema, pleural effusion and high case fatality. Previously, we demonstrated that Andes Virus (ANDV) infection caused up-regulation of vascular endothelial growth factor (VEGF) and concomitant downregulation of the cellular adhesion molecule VE-cadherin leading to increased permeability. Analyses of human HPS-patient sera have further demonstrated increased circulating levels of VEGF. Here we investigate the impact of a small molecule antagonist of the VEGF receptor 2 (VEGFR-2) activation in vitro, and overall impact on survival in the Syrian hamster model of HPS.
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Small Interfering RNA Inhibition of Andes Virus Replication
PloS one, 2014Co-Authors: Cheng-feng Chiang, Cesar G. Albariňo, Christina F. SpiropoulouAbstract:Andes Virus (ANDV) is the most common causative agent of hantaVirus pulmonary syndrome (HPS) in the Americas, and is the only hantaVirus associated with human-to-human transmission. Case fatality rates of ANDV-induced HPS are approximately 40%. There are currently no effective vaccines or antivirals against ANDV. Since HPS severity correlates with viral load, we tested small interfering RNA (siRNA) directed against ANDV genes as a potential antiviral strategy. We designed pools of 4 siRNAs targeting each of the ANDV genome segments (S, M, and L), and tested their efficacy in reducing viral replication in vitro. The siRNA pool targeting the S segment reduced viral transcription and replication in Vero-E6 cells more efficiently than those targeting the M and L segments. In contrast, siRNAs targeting the S, M, or L segment were similar in their ability to reduce viral replication in human lung microvascular endothelial cells. Importantly, these siRNAs inhibit ANDV replication even if given after infection. Taken together, our findings indicate that siRNAs targeting the ANDV genome efficiently inhibit ANDV replication, and show promise as a strategy for developing therapeutics against ANDV infection.
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Host mTORC1 Signaling Regulates Andes Virus Replication
Journal of Virology, 2013Co-Authors: Steve Mcnulty, Mariagrace Flint, Stuart T. Nichol, Christina F. SpiropoulouAbstract:HantaVirus pulmonary syndrome (HPS) is a severe respiratory disease characterized by pulmonary edema, with fatality rates of 35 to 45%. Disease occurs following infection with pathogenic New World hantaViruses, such as Andes Virus (ANDV), which targets lung microvascular endothelial cells. During replication, the Virus scavenges 5'-m(7)G caps from cellular mRNA to ensure efficient translation of viral proteins by the host cell cap-dependent translation machinery. In cells, the mammalian target of rapamycin (mTOR) regulates the activity of host cap-dependent translation by integrating amino acid, energy, and oxygen availability signals. Since there is no approved pharmacological treatment for HPS, we investigated whether inhibitors of the mTOR pathway could reduce hantaVirus infection. Here, we demonstrate that treatment with the FDA-approved rapamycin analogue temsirolimus (CCI-779) blocks ANDV protein expression and virion release but not entry into primary human microvascular endothelial cells. This effect was specific to viral proteins, as temsirolimus treatment did not block host protein synthesis. We confirmed that temsirolimus targeted host mTOR complex 1 (mTORC1) and not a viral protein, as knockdown of mTORC1 and mTORC1 activators but not mTOR complex 2 components reduced ANDV replication. Additionally, primary fibroblasts from a patient with tuberous sclerosis exhibited increased mTORC1 activity and increased ANDV protein expression, which were blocked following temsirolimus treatment. Finally, we show that ANDV glycoprotein Gn colocalized with mTOR and lysosomes in infected cells. Together, these data demonstrate that mTORC1 signaling regulates ANDV replication and suggest that the hantaVirus Gn protein may modulate mTOR and lysosomal signaling during infection, thus bypassing the cellular regulation of translation.
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Andes Virus disrupts the endothelial cell barrier by induction of vascular endothelial growth factor and downregulation of ve cadherin
Journal of Virology, 2010Co-Authors: Punya Shrivastavaranjan, Pierre E Rollin, Christina F. SpiropoulouAbstract:HantaVirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) are severe diseases associated with hantaVirus infection. High levels of Virus replication occur in microvascular endothelial cells but without a Virus-induced cytopathic effect. However, Virus infection results in microvascular leakage, which is the hallmark of these diseases. VE-cadherin is a major component of adherens junctions, and its interaction with the vascular endothelial growth factor (VEGF) receptor, VEGF-R2, is important for maintaining the integrity of the endothelial barrier. Here we report that increased secreted VEGF and concomitant decreased VE-cadherin are seen at early times postinfection of human primary lung endothelial cells with an HPS-associated hantaVirus, Andes Virus. Furthermore, active Virus replication results in increased permeability and loss of the integrity of the endothelial cell barrier. VEGF binding to VEGF-R2 is known to result in dissociation of VEGF-R2 from VE-cadherin and in VE-cadherin activation, internalization, and degradation. Consistent with this, we showed that an antibody which blocks VEGF-R2 activation resulted in inhibition of the Andes Virus-induced VE-cadherin reduction. These data implicate Virus induction of VEGF and reduction in VE-cadherin in the endothelial cell permeability seen in HPS and suggest potential immunotherapeutic targets for the treatment of the disease.
