Roles of Macrophages in Immunity to Ranavirus, and in Viral Persistence and Dissemination

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Macrophages are a type of immune cells that are indispensable to vertebrate host immunity and homeostasis. Thus, they often become targets of viral pathogens as a means of immune evasion and mechanisms of dissemination. Ranaviruses, which infect amphibians, are increasing in both prevalence and in the range of host species infected. This raises pressing concerns for biodiversity and aquaculture, and poses fundamental issues related to evolution of host/pathogen interactions. Indeed, the remarkable emerging features of ranavirus infections appear intimately linked to controlling immune responses in many host species, and growing evidence hints at macrophages as critical for their infection strategy. The goal of this research is to elucidate the complex roles of distinct amphibian adult and tadpole macrophage populations in orchestrating host immune defenses against ranavirus, while serving as reservoirs for ranavirus immune evasion and dissemination. The hypothesis addressed is that the ranavirus Frog virus 3 (FV3) targets macrophages to evade host immune defenses towards viral persistence and dissemination in the frog Xenopus laevis. The specific aims are to determine the phenotypes and fates of tadpole and adult macrophage subsets targeted by FV3; define molecular and cellular interactions of FV3 with tadpole and adult macrophages; and elucidate the roles of macrophages in host defenses and asymptomatic infections. The work will involve training of students at all levels, as well as public engagement through development of interactive programs at a local science museum. The researcher will also continue to develop and manage a resource for Xenopus immunobiology reagents and training. Macrophages are both at the forefront of immune defenses against ranavirus (Iridoviridae) pathogens and part of ranavirus infection strategies, which is unique among large DNA viruses. The dramatic worldwide increases in ranges of populations and species infected raise alarming concerns for biodiversity and aquaculture, and pose fundamental issues related to evolution of host/pathogen interactions. The overall goal of this proposed research is to elucidate the complex roles of distinct amphibian adult and tadpole macrophage populations in orchestrating host immune defenses, while serving as reservoirs for immune evasion and dissemination. The project will take advantage of the extensively characterized Xenopus/FV3 model system that permits reverse genetic as well as mechanistic experimental approaches from the molecular and cellular levels to the whole organism level. The guiding hypothesis is that FV3 targets amphibian macrophages to evade host immune defenses towards viral persistence and dissemination. The specific aims are (1) to determine the phenotypes and fates of larval and adult macrophage subsets targeted by FV3; (2) to define molecular and cellular interactions of FV3 with larval and adult macrophages; and (3) to elucidate the roles of macrophages in host defenses and asymptomatic infections. These aims will utilize technologies developed during the past NSF funding period, including RNAi-based and CRISPR/Cas9-mediated loss-of-function by transgenesis; traceable recombinant FV3 and FV3 knockout mutants deficient for virulence/immunomodulatory genes; adoptive macrophage transfer into inbred recipients; Xenopus macrophage cultures; macrophage depletion; and the use of specific Abs against Xenopus macrophages and FV3.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.