CAREER: Investigating Host Response in the Pathogenesis of FV3 (Ranavirus sp) in Wood Frogs, Rana sylvatica (Lithobates sylvaticus)

世界范围内的两栖动物正在经历前所未有的数量减少，一些科学家怀疑地球是否正处于第六次大灭绝的开始。传染病是两栖动物生存的重大威胁。蛙病毒病是由蛙病毒3型（FV3）引起的，在世界各地的野生和圈养两栖动物中引起疾病和死亡。野生蝌蚪感染FV3病毒往往导致种群死亡，死亡率接近100%，这一比率严重影响了未来野生青蛙种群的稳定性。如果RV3病毒进入圈养的两栖动物种群，就会危及濒危物种的繁殖计划。必须了解FV3的发病机制（感染如何在宿主中发展）才能减轻病毒病。FV3的发病机制受到许多因素的影响，包括：两栖动物对感染的免疫反应，以及与两栖动物生活史相关的环境因素，例如与作为病毒储存库（携带者）的物种共享栖息地或生活在不利条件下。该职业奖将研究林蛙中FV3的发病机制，林蛙是一种受ran病毒病暴发严重影响的本地蛙种。它侧重于青蛙对感染的反应，环境（温度变化），压力和存在载体对感染结果的影响。这项研究为基础研究提供资金，其研究结果将为野生和圈养两栖动物的管理决策提供信息。培养这一研究领域的研究生也将为下一代STEM研究人员做好准备。此外，还有一些外联活动，向公众宣传FV3和青蛙，以帮助人们了解目前对野生动物的威胁。从理论上讲，将FV3引入naïve林蛙（Rana sylvatica）种群将导致当地在5年内灭绝。研究者的中心假设是，宿主的生活史（生物学和生态学）、其免疫反应和其居住的环境将决定FV3感染的结果。PIs先前的研究发现，亚致命性FV3感染的幸存者（如绿蛙、蛤蛙）可能成为该病毒的长期携带者。因此，有绿蛙的环境可能与野生种群的蛙病毒病蝌蚪死亡率有关。拟开展的研究将确定蝌蚪感染的发病机制，理清（假定的）抗性物种（绿蛙）和高度易感物种（林蛙）之间的关系；确定个体在感染后是否会出现长期携带者；描述成年林蛙对FV3感染的细胞和/或体液免疫反应；并确定亚临床疾病是否对再次感染提供保护。这项工作将涉及野生捕获的、实验室饲养的蝌蚪和成虫的实验感染，LD50剂量的测定，受影响动物的组织病理学检查，应用免疫组织化学和分子标记（原位杂交）技术检测目标细胞/组织，细胞和体液免疫反应的血液学评估，以及皮质类固醇对这些反应的影响的测定。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。

Amphibians worldwide are experiencing unprecedented population losses, with some scientists wondering if the earth is at the beginning of the 6th mass extinction. Infectious diseases are a significant threat to amphibian survival. Ranavirosis is caused by Frog Virus 3 (FV3), and causes disease and death in wild and captive amphibians worldwide. FV3 infections of wild tadpoles often result in die-offs of populations reaching close to 100% mortality, a rate that significantly impacts the stability of future wild frog populations. If RV3 moves into captive amphibian populations, it jeopardized breeding programs of endangered species. There must be an understanding of FV3’s pathogenesis (how infection develops in the host) to mitigate ranavirosis. Pathogenesis of FV3 is influenced by many things including: the amphibian’s immune response to infection, and environmental factors linked to the amphibian’s life history, such as sharing habitat with species that serve as virus reservoirs (carriers) or living under unfavorable conditions. This CAREER award will investigate the pathogenesis of FV3 in wood frogs, a native frog species severely affected by outbreaks of ranavirosis. It focuses on the frog’s response to infection, the effects of environment (temperature changes), stress, and presence carriers on infection outcomes. This research funds basic research whose findings will inform decisions in management of wild and captive amphibians. Training of graduate students in this research area will also prepare the next generation of STEM researchers for the workforce. In addition, there are outreach activities to inform the general public about FV3 and frogs to help educate people about current threats to wildlife.TECHNICAL OVERVIEWIt is theorized that FV3 introduction into a naïve population of wood frogs (Rana sylvatica) will result in local extinction within 5 years. The investigator’s central hypothesis is that the life history (biology and ecology) of the host, its immune response, and the environment it inhabits will determine the outcome of FV3 infection. The PIs previous work found that survivors of sublethal FV3 infections (like green frogs, Rana clamitans) may become long-term carriers of the virus. Thus, environments with green frogs may be associated with ranavirosis tadpole mortalities in wild populations. The proposed research will determine the pathogenesis of infection in tadpoles, disentangle the relationship between (putatively) resistant species (green frogs) and highly susceptible ones (wood frogs); determine if long-term carriers develop when individuals survive infection; characterize the cellular and/or humoral immune response to FV3 infection in adult wood frogs; and determine whether subclinical disease provides protection to re-infection. The work will involve experimental infections of wild-caught, lab-raised, tadpoles and adults, determination of LD50 doses, histopathological examination of affected animals, application of immunohistochemical and molecular labeling (in situ hybridization) techniques to detect target cells/tissues, hematological evaluation of cellular and humoral immune responses, and determination of the effect of corticosteroids on those responses.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.