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.
