Development Of A Flow Cytometry Assay To Study Monkeypox Virus Infection And Immune Response Regulation in Nonhuman Primates

Abstract

Monkeypox virus (MPXV) is divided into two clades, Congo Basin and West African. Congo Basin MPXV infection is associated with severe symptoms and fatality of up to 10 percent in humans; whereas, West African MPXV infection is associated with less severe symptoms. To study infection and differential immune response regulation among virus isolates from Congo Basin and West African clades in nonhuman primates, a flow cytometry assay was developed. Annexin V, a protein used to detect apoptosis previously did not permit fixation and permeabilization for intracellular measurements, such as pathogen-infected cells. The method developed permits measurements of Annexin V apoptosis combined with intracellular staining. Reagents compatible with Annexin V were identified for fixation/permeabilization, and the need for calcium in intracellular assay steps was determined. The combined Annexin V/intracellular method described can be used to study disease pathogenesis of many BSL-3 or BSL-4 pathogens in nonhuman primates.