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

项目总结 T‘Ho病毒是一种特性不佳的黄病毒，最近在致倦库蚊中发现。 墨西哥的尤卡坦半岛。T‘Ho病毒的基因组已完成全序列测定，但仍未恢复分离株 哺乳小鼠脑接种或在脊椎动物或蚊子细胞培养中分离病毒。基因组序列 比对显示，已知的与T‘Ho病毒最接近的近亲是蚊子传播的黄病毒。 与危及生命的人类疾病有关。Rocio病毒是BSL-3病毒的病原体，是已知的最近的亲戚， 其次是伊尔赫斯病毒、圣路易斯脑炎、日本脑炎和西尼罗河病毒。因为T‘Ho病毒是最 它与已知的人类病原体密切相关，也可能是人类疾病的原因之一。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，以确定有能力的脊椎动物宿主和媒介物种。执行这些任务的能力 由于无法获得分离物，实验受到严重限制。为了解决这个问题，重组技术 将被用来制造可用于T‘Ho病毒研究和诊断的传染性病毒。第一个目标 目的是制备重组T‘Ho病毒，并对其体外宿主范围和复制动力学进行研究。基于ITS 它与在蚊子和脊椎动物宿主之间循环的黄病毒有着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都可以复制。第二个目标是创建一个 可用于BSL-2实验室检测T‘Ho病毒中和抗体的嵌合病毒。 由于T‘Ho病毒与几种BSL-3病原体关系密切，它最终可能被归类为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，a 嵌合病毒将通过取代BSL-2病原体寨卡病毒的主要结构蛋白基因而产生， 与T‘Ho病毒的相应区域结合，产生一种可用于BSL-2实验室的病毒。它是 假设上述基因交换在功能上是兼容的，并将产生嵌合体 在脊椎动物细胞单层中形成斑块的病毒。拟议的研究为许多 未来的实验。脊椎动物和蚊子可以实验接种重组病毒 以确定T‘HO病毒的合格宿主和媒介，从而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，它需要活病毒。嵌合病毒 可在BSL-3设施不可用时使用。黄病毒以其血清学交叉反应而闻名；因此， T‘Ho病毒的抗体很可能与其他黄病毒结合并中和。这增加了一种可能性，即 一些人类和脊椎动物，以前在拉丁美洲的血清学调查中被PRNT检测过 含有抗T‘Ho病毒抗体，但被误诊。重要的是有传染性的T‘Ho病毒，所以 可纳入PRNTS，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.