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

世界各地的两栖动物正在经历前所未有的人口损失，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（FV 3）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。 野生蝌蚪的FV 3感染通常导致种群的死亡率接近100%，这一比率显著影响了未来野生青蛙种群的稳定性。如果RV 3进入圈养的两栖动物种群，它会危及濒危物种的繁殖计划。必须了解FV 3的发病机制（感染如何在宿主中发展）以减轻Ranavirisis。FV 3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）或生活在不利条件下的物种共享栖息地。这个职业奖将调查FV 3在林蛙中的发病机制，林蛙是一种严重受蛙病毒病爆发影响的本地青蛙物种。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。在这一研究领域的研究生培训也将为下一代STEM研究人员做好准备。此外，还开展了宣传活动，向公众介绍FV 3和青蛙，以帮助人们了解当前野生动物面临的威胁。技术概述理论上，FV 3引入到一个天真的树蛙（Rana sylvatica）种群中将导致当地在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）、其免疫应答以及其所居住的环境将决定FV 3感染的结果。PI先前的工作发现，亚致死性FV 3感染的幸存者（如绿色青蛙，蛙）可能成为病毒的长期携带者。因此，绿色青蛙的环境可能与野生种群中的蛙病毒病蝌蚪死亡率有关。拟议的研究将确定蝌蚪感染的发病机制，解开（腐殖酸）抗性物种（绿色青蛙）和高度敏感的物种（林蛙）之间的关系;确定是否长期运营商发展时，个人生存感染;表征的细胞和/或体液免疫反应的FV 3感染成年林蛙;并确定亚临床疾病是否提供保护，以防止再次感染。这项工作将涉及野生捕获、实验室饲养的蝌蚪和成虫的实验感染、LD 50剂量的测定、受影响动物的组织病理学检查、免疫组织化学和分子标记的应用（原位杂交）技术来检测靶细胞/组织，细胞和体液免疫应答的血液学评价，该奖项反映了NSF的法定使命，并被认为是值得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的支持。

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.