scholarly journals Macropinocytosis and Clathrin-Dependent Endocytosis Play Pivotal Roles for the Infectious Entry of Puumala Virus

2019 ◽  
Author(s):  
Sandy Bauherr ◽  
Filip Larsberg ◽  
Annett Petrich ◽  
Hannah Sabeth Sperber ◽  
Victoria Klose ◽  
...  
Keyword(s):  
Rna Interference ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Case Fatality ◽  
Hemorrhagic Fever ◽  
Puumala Virus ◽  
Virus Species ◽  
Global Public Health ◽  
Emerging Pathogens ◽  
Cellular Mechanisms

AbstractViruses from the taxonomic familyHantaviridaeare encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with case fatalities of up to 50%. Here we comprehensively investigated entry of the Old-World Hantavirus, Puumala virus (PUUV), into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are significantly reduced. We demonstrated that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, is able to decrease PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV virus particles and showed co-localization of stained virions and markers of macropinocytic uptake. Cells treated with lysosomotrophic agents were shown to exhibit an increased resistance to infection, confirming previous data suggesting that a low pH-dependent step is involved in PUUV infection. Finally, we observed a significant increase in the fluid-phase uptake of cell infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis.Author SummaryTheHantaviridaefamily comprises a very diverse group of virus species and is considered an emerging global public health threat. Human pathogenic hantaviruses are primarily rodent-borne. Zoonosis is common with more than 150,000 annually registered cases and a case fatality index of up to 50%. Individual hantavirus species differ significantly in terms of their pathogenicity, but also their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV virions into mammalian cells. We showed that both, clathrin-mediated endocytosis and macropinocytosis, are cellular pathways exploited by the virus to establish productive infections and demonstrated that pharmacological inhibition of macropinocytosis or its targeted knockdown using RNA interference significantly reduced viral infections. We also found indications for an increase of macropinocytic uptake upon PUUV infections, suggesting that the virus triggers specific cellular mechanisms in order to promote its own internalization and facilitate infections.

scholarly journals Macropinocytosis and Clathrin-Dependent Endocytosis Play Pivotal Roles for the Infectious Entry of Puumala Virus

2020 ◽  
Vol 94 (14) ◽  
Author(s):  
Sandy Bauherr ◽  
Filip Larsberg ◽  
Annett Petrich ◽  
Hannah Sabeth Sperber ◽  
Victoria Klose-Grzelka ◽  
...  
Keyword(s):  
Rna Interference ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Case Fatality ◽  
Hemorrhagic Fever ◽  
Puumala Virus ◽  
Virus Species ◽  
Global Public Health ◽  
Emerging Pathogens ◽  
The Family

ABSTRACT Viruses from the family Hantaviridae are encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with case fatality rates of up to 50%. Here, we comprehensively investigated entry of the Old World hantavirus Puumala virus (PUUV) into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are greatly reduced. We demonstrate that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, decreases PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV particles and show colocalization of stained virions and markers of macropinosomes. Finally, we report a significant increase in the fluid-phase uptake of cells infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis. IMPORTANCE The family Hantaviridae comprises a diverse group of virus species and is considered an emerging global public health threat. Individual hantavirus species differ considerably in terms of their pathogenicity but also in their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV into mammalian cells. We show that both clathrin-mediated endocytosis and macropinocytosis are cellular pathways exploited by the virus to establish productive infections and demonstrate that pharmacological inhibition of macropinocytosis or a targeted knockdown using RNA interference significantly reduced viral infections. We also found indications of an increase of macropinocytic uptake upon PUUV infection, suggesting that the virus triggers specific cellular mechanisms in order to stimulate its own internalization, thus facilitating infection.

scholarly journals Ferrets Infected with Bundibugyo Virus or Ebola Virus Recapitulate Important Aspects of Human Filovirus Disease

2016 ◽  
Vol 90 (20) ◽  
pp. 9209-9223 ◽  
Author(s):  
Robert Kozak ◽  
Shihua He ◽  
Andrea Kroeker ◽  
Marc-Antoine de La Vega ◽  
Jonathan Audet ◽  
...  
Keyword(s):  
Animal Model ◽  
Ebola Virus ◽  
Virus Transmission ◽  
Case Fatality ◽  
Small Animal ◽  
Hemorrhagic Fever ◽  
Transmission Model ◽  
Global Public Health ◽  
Small Animal Model ◽  
Content Type

ABSTRACTBundibugyo virus (BDBV) is the etiological agent of a severe hemorrhagic fever in humans with a case-fatality rate ranging from 25 to 36%. Despite having been known to the scientific and medical communities for almost 1 decade, there is a dearth of studies on this pathogen due to the lack of a small animal model. Domestic ferrets are commonly used to study other RNA viruses, including members of the orderMononegavirales. To investigate whether ferrets were susceptible to filovirus infections, ferrets were challenged with a clinical isolate of BDBV. Animals became viremic within 4 days and succumbed to infection between 8 and 9 days, and a petechial rash was observed with moribund ferrets. Furthermore, several hallmarks of human filoviral disease were recapitulated in the ferret model, including substantial decreases in lymphocyte and platelet counts and dysregulation of key biochemical markers related to hepatic/renal function, as well as coagulation abnormalities. Virological, histopathological, and immunohistochemical analyses confirmed uncontrolled BDBV replication in the major organs. Ferrets were also infected with Ebola virus (EBOV) to confirm their susceptibility to another filovirus species and to potentially establish a virus transmission model. Similar to what was seen with BDBV, important hallmarks of human filoviral disease were observed in EBOV-infected ferrets. This study demonstrates the potential of this small animal model for studying BDBV and EBOV using wild-type isolates and will accelerate efforts to understand filovirus pathogenesis and transmission as well as the development of specific vaccines and antivirals.IMPORTANCEThe 2013-2016 outbreak of Ebola virus in West Africa has highlighted the threat posed by filoviruses to global public health. Bundibugyo virus (BDBV) is a member of the genusEbolavirusand has caused outbreaks in the past but is relatively understudied, likely due to the lack of a suitable small animal model. Such a model for BDBV is crucial to evaluating vaccines and therapies and potentially understanding transmission. To address this, we demonstrated that ferrets are susceptible models to BDBV infection as well as to Ebola virus infection and that no virus adaptation is required. Moreover, these animals develop a disease that is similar to that seen in humans and nonhuman primates. We believe that this will improve the ability to study BDBV and provide a platform to test vaccines and therapeutics.

scholarly journals Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

2012 ◽  
Vol 20 (2) ◽  
pp. 218-226 ◽  
Author(s):  
R. L. Brocato ◽  
M. J. Josleyn ◽  
V. Wahl-Jensen ◽  
C. S. Schmaljohn ◽  
J. W. Hooper
Keyword(s):  
Dna Vaccine ◽  
Neutralizing Antibodies ◽  
High Titer ◽  
Disease Model ◽  
Case Fatality ◽  
Hemorrhagic Fever ◽  
Puumala Virus ◽  
M Gene ◽  
Reading Frame ◽  
Andes Virus

ABSTRACTPuumala virus (PUUV) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). Although PUUV-associated HFRS does not result in high case-fatality rates, the social and economic impact is considerable. There is no licensed vaccine or specific therapeutic to prevent or treat HFRS. Here we report the synthesis of a codon-optimized, full-length M segment open reading frame and its cloning into a DNA vaccine vector to produce the plasmid pWRG/PUU-M(s2). pWRG/PUU-M(s2) delivered by gene gun produced high-titer neutralizing antibodies in hamsters and nonhuman primates. Vaccination with pWRG/PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses. Unexpectedly, vaccination protected hamsters in a lethal disease model of Andes virus (ANDV) in the absence of ANDV cross-neutralizing antibodies. This is the first evidence that an experimental DNA vaccine for HFRS can provide protection in a hantavirus lethal disease model.

scholarly journals Immunogenic Properties of MVs Containing Structural Hantaviral Proteins: An Original Study

Pharmaceutics ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 93
Author(s):  
Layaly Shkair ◽  
Ekaterina Evgenevna Garanina ◽  
Ekaterina Vladimirovna Martynova ◽  
Alena Igorevna Kolesnikova ◽  
Svetlana Sergeevna Arkhipova ◽  
...  
Keyword(s):  
Hemorrhagic Fever ◽  
Original Study ◽  
Puumala Virus ◽  
Global Public Health ◽  
Public Health Threat ◽  
Delivery Vehicles ◽  
Cellular Immune ◽  
Immunogenic Properties

Hemorrhagic fever with renal syndrome (HFRS) is an emerging infectious disease that remains a global public health threat. The highest incidence rate is among zoonotic disease cases in Russia. Most cases of HFRS are reported in the Volga region of Russia, which commonly identifies the Puumala virus (PUUV) as a pathogen. HFRS management is especially challenging due to the lack of specific treatments and vaccines. This study aims to develop new approaches for HFRS prevention. Our goal is to test the efficacy of microvesicles (MVs) as PUUV nucleocapsid (N) and glycoproteins (Gn/Gc) delivery vehicles. Our findings show that MVs could deliver the PUUV N and Gn/Gc proteins in vitro. We have also demonstrated that MVs loaded with PUUV proteins could elicit a specific humoral and cellular immune response in vivo. These data suggest that an MV-based vaccine could control HFRS.

scholarly journals Mammalian synthetic biology for studying the cell

2016 ◽  
Vol 216 (1) ◽  
pp. 73-82 ◽  
Author(s):  
Melina Mathur ◽  
Joy S. Xiang ◽  
Christina D. Smolke
Keyword(s):  
Synthetic Biology ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Dynamic Control ◽  
Model Systems ◽  
Cellular Mechanisms ◽  
Cellular Processes ◽  
Regulatory Processes ◽  
Multicellular Interactions

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology.

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