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

TâHo病毒的表型特征及其血清学诊断工具的开发

• 批准号: 10618135
• 负责人: Bradley J Blitvich
• 金额: $ 7.65万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-05 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10618135
• 关键词: Address; Aedes; Americas; Anopheles Genus; Antibodies; Applications Grants; Arboviruses; Binding; Biological Assay; Birds; Brain; Catalytic RNA; Cell Culture Techniques; Cell Line; Cells; Classification; Clinical; Collection; Complementary DNA; Culex (Genus); Culicidae; DNA; Data; Deposition; Detection; Diagnosis; Differential Diagnosis; Disease; Equus caballus; Flavivirus; Flavivirus Infections; Foundations; Future; Gene Proteins; Genetic; Genome; Genomics; Geographic Distribution; Hamsters; Hepatitis Delta Virus; Human; In Vitro; Infection; Insecta; Investigation; Japanese Encephalitis; Japanese encephalitis virus; Kinetics; Laboratories; Latin America; Length; Life; Measures; Membrane Proteins; Mexico; Monitor; Morphology; Mus; Neutralization Tests; Phenotype; Phylogenetic Analysis; Poly A; Polyadenylation; Recombinants; Research; Research Personnel; Rodent; Sequence Alignment; Serodiagnoses; Serology; Seroprevalences; Signal Transduction; Simian virus 40; St. Louis Encephalitis; St. Louis Encephalitis Virus; Structural Protein; Techniques; Technology; Testing; Ticks; Transfection; Vertebrates; Viral; Virus; Virus Replication; West Nile virus; Zika Virus; biosafety level 3 facility; cross reactivity; design; env Gene Products; experimental study; human disease; human pathogen; in vivo; insight; monolayer; neutralizing antibody; nonhuman primate; outcome prediction; pathogen; promoter; recombinant virus; serosurveillance; suckling; tool development; transmission process; vaccine development; vector; whole genome

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.

项目摘要 T'HO病毒是最近在Culex quinquefasciatus蚊子中发现的一种特征性的黄病毒。 墨西哥的尤卡坦半岛。 T'HO病毒的基因组已完全测序，但没有通过 哺乳小鼠脑接种或通过脊椎动物或蚊子细胞培养物中的病毒分离。基因组序列 一致性表明，t'ho病毒的最接近的亲属是蚊子传播的黄病毒 与威胁生命的人类疾病有关。 Rocio病毒是一种BSL-3病原体，是最接近的亲戚， 其次是伊拉斯（Ilhéus），圣路易斯脑炎，日本脑炎和西尼罗河病毒。因为t'ho病毒是最多的 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 需要进行研究以调查此问题。还需要在体内执行的重要需求 实验感染以鉴定有效的脊椎动物宿主和载体物种。执行这些的能力 实验受到分离株不可用的严格限制。为了解决这个问题，重组技术 将用于创建可用于T'HO病毒研究和诊断的传染病。第一个目标 是为了产生重组病毒，并表征其体外宿主范围和复制动力学。基于它的 与蚊子和脊椎动物宿主之间循环循环的黄病毒的密切系统发育关系，它是 假设T'HO病毒在蚊子和脊椎动物细胞中均复制。第二个目标是创建一个 可以在BSL-2实验室中用于检测中和T'HO病毒抗体的嵌合病毒。 由于T'HO病毒与几种BSL-3病原体密切相关，因此最终可以将其归类为BSL-3药物。 但是，许多Arbovirus实验室，尤其是拉丁美洲的实验室，缺乏BSL-3的设施。因此， 嵌合病毒将通过取代Zika病毒的主要结构蛋白基因（BSL-2病原体）来产生 与T'HO病毒相应的区域，可在BSL-2实验室中产生一种病毒。这是 假设合理的遗传交换在功能上是兼容的，并且会产生嵌合 在脊椎动物细胞单层中形成斑块的病毒。拟议的研究为许多人提供了基础 未来的实验。脊椎动物和蚊子可以通过重组实验接种 病毒以识别T'HO病毒的有能力的储层宿主和向量，从而洞悉其传输周期。 重组病毒将允许研究人员监测墨西哥和其他地方的人类和脊椎动物 在美洲，用于通过牙菌斑减少中和试验（PRNT）抗体的抗体。 prnt是金 - 用于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，可以使用。黄病毒以其血清学交叉反应性而闻名。因此， 对T'HO病毒的抗体可能会结合并中和其他黄病毒。这增加了 一些以前在拉丁美洲的血清学研究中通过PRNT测试的人类和脊椎动物动物 含有与病毒的抗体，但被诊断出来。重要的是有传染性病毒可用 它可以包含在PRNT中，并在差异诊断中考虑。