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Principal Investigator: Luis J Sigal
Organization: THOMAS JEFFERSON UNIVERSITY
Fiscal Year: 2022
Award: $195,000
Funding agency: National Institute of Allergy and Infectious Diseases
Summary
Prevention of morbidity and mortality from viral infections and of complications from live-virus vaccine
immunizations requires the identification of genes that confer natural resistance to viruses. Ectromelia virus
(ECTV), the agent of mousepox, is a natural pathogen of the mouse. When ECTV is inoculated into the footpad
of mice, it rapidly spreads to the draining lymph node (dLN) through afferent lymphatics. After replicating in the
dLN, ECTV spreads to the bloodstream through efferent lymphatics, and through the blood, it reaches its main
target organs, the liver and the spleen. In susceptible strain of mice such as BALB/c, the replication in the liver
is massive, reaching ~109 plaque forming units (pfu)/g at 7 days post infection (dpi). This results in liver necrosis
and death. Yet, in C57BL/6 (B6) and other mousepox resistant mouse strains, ECTV also becomes systemic but
the viral load in the liver at 7 dpi reaches only ~104 pfu/g and ECTV is cleared by a strong immune response
without major signs of disease.
We have been studying the mechanisms whereby B6 mice resist mousepox for many years. We have found
that a main cause of resistance is a highly choreographed innate immune response in the dLN which delays viral
spread and preponderantly involves inflammatory monocytes (iMOs) which must produce and sense Type I
interferon (IFN-I). The Specific Aim of this grant is to discover new genes involved in iMO-mediated protective
anti-viral innate immunity through the generation of mice in which iMOs specifically lack interferon stimulated
genes (ISGs). In Subaim A, we will produce mice with floxed alleles in 5 ISGs that we have identified through
RNA-Seq. These ISGs where upregulated in vivo to high levels and at least three-fold from naïve controls in
infected and/or uninfected iMOs in an IFN-I dependent manner. They are not well-known but are conserved in
humans. To produce these mice, we will use the method “Oviductal Nucleic Acids Delivery (i-GONAD)” that
delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. We have successfully
established iGONAD in our lab. This allows us to produce mutant mice rapidly and at a low cost. In Subaim B:
Floxed mice will be bred with Lyz2-Cre mice which deletes floxed genes in the monocytes/macrophage linage.
We will use these mice to determine whether the genes are specifically required for monocytes/macrophage
development and for iMO-mediated resistance to mousepox. We predict that at least some of these genes will
be critical for resistance to mousepox. Moreover, we speculate that these genes will be critical for resistance to
a variety of pathogens and also important for the resistance of humans to viral infections and for lack of
complications after live-vaccine immunization. This could be tested in the future.
Terms: <Acute><Affect><Animal Model><Animal Models and Related Studies><Antiviral resistance><Antiviral resistant><Attenuated Vaccines><BALB C Mouse><BALB/c><Blood><Blood Circulation><Blood Reticuloendothelial System><Blood monocyte><Bloodstream><CRISPR><CRISPR/Cas system><CXCL9><CXCL9 gene><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Cessation of life><Chemotactic Cytokines><Circulation><Clustered Regularly Interspaced Short Palindromic Repeats><Cytotoxic cell><Death><Dendritic Cells><Development><Disease><Disorder><Ectromelia virus><Electroporation><Embryo><Embryonic><Fallopian Tubes><Future><Gamma interferon><Gene Transcription><Generations><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Goals><Grant><Health><Hematopoietic><Homologous Chemotactic Cytokines><Human><Humig><IFN><IFN-Gamma><IFN-alphaR><IFN-g><IFN-αR><IFN-γ><IFNAR><IFNAR1><IFNAR1 gene><IFNG><IFNγ><Immune Interferon><Immune response><Immunization><Immunologic Sensitization><Immunologic Stimulation><Immunological Sensitization><Immunological Stimulation><Immunological response><Immunostimulation><In Situ><In Vitro><Inbred BALB C Mice><Individual><Infection><Infectious Ectromelia><Infectious Skin Diseases><Inflammatory><Inflammatory Response><Innate Immune Response><Innate Immunity><Intercrines><Interferon Alpha Receptor Complex><Interferon Beta Receptor><Interferon Gamma><Interferon Type I><Interferon Type II><Interferon-gamma><Interferons><Intracellular Communication and Signaling><K lymphocyte><Knowledge><Live-attenuated Vaccine><Liver><Liver necrosis><LoxP-flanked allele><Lymphatic><MIG Gene><Mammalian Oviducts><Marrow monocyte><Mediating><Methods><Mice><Mice Mammals><Modeling><Modern Man><Morbidity><Morbidity - disease rate><Mouse Pox><Mouse Pox Virus><Mouse Strains><Mousepox><Mousepox virus><Murine><Mus><Mutant Strains Mice><Mutation><Mφ><NK Cells><Native Immunity><Natural Immunity><Natural Killer Cells><Natural Resistance><Necrosis hepatocellular><Non-Specific Immunity><Nonspecific Immunity><Organ><Outcome><P variolae><P. variolae><P.variolae><Persons><Play><Poxvirus muris><Poxvirus variolae><Prevention><Proteins><RNA Expression><RNA Seq><RNA sequencing><RNAseq><Resistance><Ribonucleoproteins><Role><SCYB9><SIS cytokines><Salpinx><Signal Transduction><Signal Transduction Systems><Signaling><Skin><Smallpox><Smallpox Vaccine><Smallpox virus><Spleen><Spleen Reticuloendothelial System><Tamoxifen><Testing><Time><Transcription><Uterine Tubes><Vaccines><Variola><Variola major virus><Variola virus><Veiled Cells><Viral><Viral Activity><Viral Burden><Viral Diseases><Viral Function><Viral Load><Viral Load result><Viral Physiology><Virus><Virus Diseases><Work><adaptive immune response><anti-viral resistance><anti-viral resistant><biological signal transduction><chemoattractant cytokine><chemokine><conditional mutant><conditional mutation><cost><crg-10><cutaneous infection><developmental><discover genes><draining lymph node><electroporative delivery><expectation><experience><experiment><experimental research><experimental study><floxed><floxed allele><gene discovery><gene electrotransfer><gene manipulation><genetic manipulation><genetically manipulate><genetically perturb><genome mutation><hemopoietic><hepatic body system><hepatic necrosis><hepatic organ system><host response><immune system response><immunoresponse><in vivo><infected skin><interferon alpha receptor><interferon α receptor><lFN-Gamma><live vaccine><live vaccines><mRNA seq><mRNA sequencing><mRNA-seq><mRNAseq><macrophage><model of animal><model organism><monocyte><mortality><mouse mutant><naturally resistant><nucleic acid delivery><oviduct><pathogen><prevent><preventing><recruit><regional lymph node><resistant><response><skin infection><small pox><small pox vaccine><small pox virus><social role><tool><transcriptome sequencing><variola major><viral infection><virus infection><virus-induced disease>