Phenotypic characterization of T’Ho virus and the development of tools for its serologic diagnosis

项目摘要 T 'Ho病毒是一种特征不明显的黄病毒，最近在非洲大陆的致倦库蚊（Culex quinquefasciatus）中发现。 墨西哥尤卡坦半岛。对T 'Ho病毒的基因组进行了完全测序，但通过PCR未回收到分离株。 乳鼠脑接种或通过在脊椎动物或蚊子细胞培养物中的病毒分离。基因组序列 比对结果显示，T 'Ho病毒已知的最接近的亲属是蚊子传播的黄病毒 与危及生命的人类疾病有关。Rocio病毒，一种BSL-3病原体，是已知的最接近的亲戚， 其次是伊列乌斯病毒、圣路易斯脑炎、日本脑炎和西尼罗河病毒。因为T 'Ho病毒是 与已知的人类病原体密切相关，它也可能是人类疾病的原因。临床和血清学 需要进行研究来调查这个问题。还有一个重要的需要是在体内进行 实验感染，以确定主管脊椎动物宿主和载体物种。执行这些任务的能力 分离物的不可用严重限制了实验。为了解决这个问题，重组技术 将被用于创造可用于T 'Ho病毒研究和诊断的传染性病毒。第一个目标 制备重组T 'Ho病毒并表征其体外宿主范围和复制动力学。基于其 与在蚊子和脊椎动物宿主之间循环的黄病毒有密切的系统发育关系， 假设T 'Ho病毒在蚊子和脊椎动物细胞中复制。第二个目标是建立一个 嵌合病毒，可用于BSL-2实验室检测T 'Ho病毒的中和抗体。 由于T 'Ho病毒与几种BSL-3病原体密切相关，因此最终可将其归类为BSL-3病原体。 然而，许多虫媒病毒实验室，特别是拉丁美洲的实验室，缺乏BSL-3设施。因此 嵌合病毒将通过取代寨卡病毒（一种BSL-2病原体）的主要结构蛋白基因， 与T 'Ho病毒的相应区域连接，产生可用于BSL-2实验室的病毒。是 假设上述基因交换在功能上是相容的，并将产生嵌合体。 在脊椎动物细胞单层中形成噬斑的病毒。拟议的研究为许多研究提供了基础。 未来的实验脊椎动物和蚊子可以用重组体进行实验性接种 病毒，以确定主管水库主机和载体的T 'Ho病毒，提供深入了解其传播周期。 这种重组病毒将使研究人员能够监测墨西哥和其他地方的人类和脊椎动物 在美洲，通过空斑减少中和试验（PRNT）检测T 'Ho病毒抗体。PRNT是黄金- 诊断黄病毒感染的标准血清学技术，需要活病毒。嵌合病毒 当BSL-3设施不可用时可使用。黄病毒以其血清学交叉反应性而闻名;因此， T 'Ho病毒的抗体可能结合并中和其它黄病毒。这就提出了一种可能性， 一些人类和脊椎动物先前在拉丁美洲的血清学研究中通过PRNT进行了检测 含有T 'Ho病毒的抗体但被误诊了重要的是，传染性T 'Ho病毒是可用的， 它可以被包括在PRNT中并在鉴别诊断中考虑。

PROJECT SUMMARY T’Ho virus is a poorly characterized flavivirus recently discovered in Culex quinquefasciatus mosquitoes in the Yucatan Peninsula of Mexico. The genome of T’Ho virus was fully sequenced but an isolate was not recovered by suckling mouse brain inoculation or by virus isolation in vertebrate or mosquito cell cultures. Genome sequence alignments revealed that the closest known relatives of T’Ho virus are mosquito-transmitted flaviviruses associated with life-threatening human disease. Rocio virus, a BSL-3 pathogen, is the closest known relative, followed by Ilhéus, St. Louis encephalitis, Japanese encephalitis and West Nile viruses. Because T’Ho virus is most closely related to known human pathogens, it too could be a cause of human disease. Clinical and serological studies need to be performed to investigate this issue. There is also an important need to perform in vivo experimental infections to identify competent vertebrate host and vector species. The ability to perform these experiments is severely restricted by the unavailability of an isolate. To address this issue, recombinant technology will be used to create infectious viruses that can be used for T’Ho virus research and diagnosis. The first objective is to generate recombinant T’Ho virus and characterize its in vitro host range and replication kinetics. Based on its close phylogenetic relationship with flaviviruses that cycle between mosquitoes and vertebrate hosts, it is hypothesized that T’Ho virus replicates in both mosquito and vertebrate cells. The second objective is to create a chimeric virus that can be used in BSL-2 laboratories for the detection of neutralizing antibodies to T’Ho virus. Because T’Ho virus is closely related to several BSL-3 pathogens, it could eventually be classified as a BSL-3 agent. However, many arbovirus laboratories, particularly those in Latin America, lack BSL-3 facilities. Therefore, a chimeric virus will be generated by substituting the major structural protein genes of Zika virus, a BSL-2 pathogen, with the corresponding region of T’Ho virus, producing a virus that can be used in BSL-2 laboratories. It is hypothesized that the aforementioned genetic exchange is functionally compatible and will generate a chimeric virus that forms plaques in vertebrate cell monolayers. The proposed studies provide the foundation for many future experiments. Vertebrate animals and mosquitoes can be experimentally inoculated with the recombinant virus to identify competent reservoir hosts and vectors of T’Ho virus, providing insight into its transmission cycle. The recombinant virus will allow researchers to monitor humans and vertebrate animals in Mexico and elsewhere in the Americas for antibodies to T’Ho virus by plaque reduction neutralization test (PRNT). The PRNT is the gold- standard serologic technique for the diagnosis of flavivirus infections and it requires live virus. The chimeric virus can be used when BSL-3 facilities are unavailable. Flaviviruses are known for their serologic cross-reactivity; thus, it is likely that antibodies to T’Ho virus can bind to and neutralize other flaviviruses. This raises the possibility that some human and vertebrate animals previously tested by PRNT in serologic investigations in Latin America contained antibodies to T’Ho virus but were misdiagnosed. It is important that infectious T’Ho virus is available so it can be included in PRNTs and considered in the differential diagnosis.