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

巨噬细胞是一种对脊椎动物宿主免疫和体内平衡至关重要的免疫细胞。因此，它们经常成为病毒病原体的目标，作为一种免疫逃避手段和传播机制。感染两栖动物的ranavirus的流行率和受感染宿主物种的范围都在增加。这引起了对生物多样性和水产养殖的紧迫关注，并提出了与宿主/病原体相互作用演变有关的基本问题。事实上，ranavirus感染的显著新特征似乎与控制许多宿主物种的免疫反应密切相关，并且越来越多的证据表明巨噬细胞对其感染策略至关重要。本研究的目的是阐明不同两栖动物成年和蝌蚪巨噬细胞群体在协调宿主免疫防御中对ranavirus的复杂作用，同时作为ranavirus免疫逃避和传播的储存库。本文提出的假设是蛙蛙病毒3 （FV3）以巨噬细胞为靶点，逃避宿主对病毒在非洲爪蟾（Xenopus laevis）体内持续传播的免疫防御。具体目的是确定FV3靶向的蝌蚪和成年巨噬细胞亚群的表型和命运；确定FV3与蝌蚪和成年巨噬细胞的分子和细胞相互作用；并阐明巨噬细胞在宿主防御和无症状感染中的作用。这项工作将包括对各级学生的培训，以及通过在当地科学博物馆开发互动项目来吸引公众参与。研究人员还将继续开发和管理非洲爪蟾免疫生物学试剂和培训资源。巨噬细胞既处于对病毒病原体免疫防御的前沿，也是病毒感染策略的一部分，这在大型DNA病毒中是独一无二的。世界范围内受感染的种群和物种的急剧增加引起了对生物多样性和水产养殖的惊人关注，并提出了与宿主/病原体相互作用演变有关的根本问题。本研究的总体目标是阐明不同两栖动物成年和蝌蚪巨噬细胞群体在协调宿主免疫防御中的复杂作用，同时作为免疫逃避和传播的储存库。该项目将利用广泛表征的Xenopus/FV3模型系统，该系统允许从分子和细胞水平到整个生物体水平的反向遗传和机械实验方法。指导假设是FV3靶向两栖动物巨噬细胞以逃避宿主对病毒持续和传播的免疫防御。具体目的是：(1)确定FV3靶向的幼虫和成体巨噬细胞亚群的表型和命运；(2)确定FV3与幼虫和成体巨噬细胞的分子和细胞相互作用；(3)阐明巨噬细胞在宿主防御和无症状感染中的作用。这些目标将利用过去NSF资助期间开发的技术，包括基于rnai和CRISPR/ cas9介导的转基因功能丧失；缺乏毒力/免疫调节基因的可追溯重组FV3和FV3敲除突变体；过继性巨噬细胞移入近交受体；爪蟾巨噬细胞培养；巨噬细胞耗竭;以及使用特异性抗体对抗爪蟾巨噬细胞和FV3。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.