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Principal Investigator: YOSHIHIRO KAWAOKA
Organization: UNIVERSITY OF WISCONSIN-MADISON
Fiscal Year: 2020
Award: $206,568
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY
Ebola viruses and other members of the filovirus family cause severe and often lethal infections. While some
progress has been made in regards to the development of experimental countermeasures and insights into the
highly pathogenic nature of these viruses, there are still many more unanswered questions, and further
advancement is needed towards the development of pan-filovirus therapeutic agents and vaccines. A significant
hurdle to research on filoviruses is the accessibility and cost associated with high-containment, biosafety level-
4 laboratories. To partially alleviate this issue, we previously established a replication-defective Ebola virus
based on the Zaire ebolavirus (EBOV) genome. This virus, which lacks the essential viral gene VP30 (termed
EBOVΔVP30), is biologically inert and safe to use outside of highly specialized BSL-4 containment. In
engineered cell lines that stably express EBOV VP30, the virus becomes replication-competent; thus it is a
perfect EBOV surrogate for in vitro research given that EBOVΔVP30 resembles authentic virus in its life cycle,
morphology, protein composition, and growth kinetics. After extensive safety testing both in cell culture and in
animal models, the CDC and the Office of Science Policy at the NIH classified EBOVΔVP30 as a BSL-2 agent
and removed the virus from Select Agent regulations. Since then, this in vitro system to study EBOV has been
requested by and distributed to several other research laboratories.
The next step to advance the EBOVΔVP30 system is to develop an EBOV VP30 transgenic animal model to
support virus replication. Previously, we generated a transgenic mouse line that expressed EBOV VP30 under
the control of the chicken beta-actin promoter (CAG). Although we were able to detect VP30 mRNA in key
organs, such as the liver, and functional VP30 protein in cells, such as fibroblasts, we were unable to detect
VP30 mRNA and functional protein in monocyte-derived macrophages, the first target of EBOV infection and a
cell type essential for virus dissemination throughout the body. Here, we propose to generate a new transgenic
mouse line with EBOV VP30 expression under the control of the CD45 promoter, a promoter specific for
expression in hematopoietic cells, including monocytes, macrophages, and dendritic cells. Once we confirm the
expression and function of VP30 in these cell types, particularly macrophages, we will cross our two different
VP30 transgenic lines (CAG and CD45) to generate a double knock-in transgenic mouse line. Once established,
we will infect these transgenic mice with mouse-adapted EBOVΔVP30 and characterize the phenotype. We will
also generate chimeric versions of EBOVΔVP30 with glycoproteins from other filoviruses and examine the
phenotype of these chimeric viruses in the transgenic mice. After safety testing, this new small animal model will
be an ideal in vivo surrogate for the authentic mouse model for EBOV infection. For the first time, a transgenic
mouse model will be available that can be used efficiently and safely outside of BSL-4 containment to examine
EBOV pathogenesis and accelerate the development and evaluation of countermeasures.
Terms: <African><Animal Model><Animal Models and Related Studies><Antiviral Agents><Antiviral Drugs><Antivirals><B220><BDBV><Bats><Belgian Congo><Biological><Blood monocyte><Breeding><Bundibugyo ebola virus><Bundibugyo ebolavirus><Bundibugyo virus><CD45><CDC><Case Fatality Rates><Cell Body><Cell Culture Techniques><Cells><Centers for Disease Control><Centers for Disease Control and Prevention><Centers for Disease Control and Prevention (U.S.)><Cessation of life><Chickens><China><Chiroptera><Collaborations><Consumption Coagulopathy><Containment><Death><Defective Hybrids><Defective Interfering Particles><Defective Interfering Viruses><Defective Viruses><Democratic Republic of the Congo><Dendritic Cells><Development><Development and Research><Disease><Disease Outbreaks><Disorder><Disseminated Intravascular Coagulation><EBOV><Ebola><Ebola virus><Engineering><Evaluation><Failure><Family><Fibroblasts><Filoviridae><Filovirus><French Guinea><Future><GP180><Gallus domesticus><Gallus gallus><Gallus gallus domesticus><Generalized Growth><Genome><Glycoproteins><Goals><Growth><Guinea><Hematopoietic><Histopathology><Human><Immune response><Immunochemical Immunologic><Immunologic><Immunological><Immunological response><Immunologically><Immunologics><In Vitro><Incomplete Viruses><Infection><Investigators><Kenya><Kinetics><Knock-in><LY5><Laboratories><Laboratory Research><Life Cycle><Life Cycle Stages><Liver><Mainland China><Marrow monocyte><Messenger RNA><Mice><Mice Mammals><Modeling><Modern Man><Morphology><Murine><Mus><NIH><National Institutes of Health><Nature><Organ><Outbreaks><PTPRC><PTPRC gene><Pathogenesis><Pathogenicity><Pathogenicity Factors><Phenotype><Property><Proteins><Public Health><R & D><R&D><RESTV><Regulation><Research><Research Personnel><Researchers><Resistance><Reston Ebola virus><Reston ebolavirus><Reston virus><Risk><SUDV><Science Policy><Sierra Leone><Spain><Sudan Ebola virus><Sudan ebolavirus><Sudan virus><System><T200><Tail><Therapeutic><Therapeutic Agents><Time><Tissue Growth><Training><Transgenic Animals><Transgenic Mice><Transgenic Organisms><United States Centers for Disease Control><United States Centers for Disease Control and Prevention><United States National Institutes of Health><Universities><Vaccines><Veiled Cells><Viral><Viral Genes><Viral Genome><Virulence Factors><Virus><Virus Replication><Wild Type Mouse><Wisconsin><ZEBOV><Zaire><Zaire Ebola virus><Zaire ebolavirus><anti-viral agents><anti-viral drugs><anti-virals><base><beta Actin><cell culture><cell type><cost><developmental><disease phenotype><ebolavirus><forest><genome editing><genomic editing><hemopoietic><hepatic body system><hepatic organ system><host response><human disease><immunoresponse><in vivo><insight><knockin><life course><mRNA><macrophage><member><model of animal><model organism><monocyte><mouse model><murine model><ontogeny><pathogenic virus><promoter><promotor><research and development><resistant><safety testing><stable cell line><therapeutic vaccine><transgenic><treatment vaccines><vaccine for the treatment><vaccine for treatment><viral multiplication><viral pathogen><viral replication><virus genome><virus multiplication><wildtype mouse><β-Actin>