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

基本信息

批准号：
10363430
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
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-05 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10363430
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 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 Gold Hamsters Hepatitis Delta Virus Human In Vitro Infection Insecta Investigation Japanese Encephalitis Japanese encephalitis virus Kinetics Laboratories Latin America Length Life Measures Mexico Monitor Morphology Mus Neutralization Tests Phenotype Phylogenetic Analysis 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 Vertebrates Viral Virus West Nile virus Zika Virus base 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 virus development whole genome

项目摘要

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.

项目概要 T’Ho 病毒是一种特征不明的黄病毒，最近在美国的致倦库蚊 (Culex quinquefasciatus) 中发现。墨西哥尤卡坦半岛的 T’Ho 病毒基因组已完成测序，但尚未发现分离株。乳鼠大脑接种或通过脊椎动物或蚊子细胞培养物中的病毒分离。比对显示，已知的 T’Ho 病毒最近亲是蚊子传播的黄病毒 Rocio 病毒是一种 BSL-3 病原体，与危及生命的人类疾病相关，是已知的最接近的亲属。其次是伊列斯脑炎、圣路易斯脑炎、日本脑炎和西尼罗河病毒，因为T'Ho病毒最多。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。需要进行研究来调查这个问题，还需要进行体内研究。实验感染以确定有能力的脊椎动物宿主和载体物种执行这些操作的能力。实验受到分离株的缺乏的严重限制。为了解决这个问题，重组技术被采用。将用于制造可用于 T'Ho 病毒研究和诊断的传染性病毒。旨在产生重组 T’Ho 病毒并根据其体外宿主范围和复制动力学进行表征。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，培养 T’Ho 病毒在蚊子和脊椎动物细胞中复制。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 病毒与多种 BSL-3 病原体密切相关，因此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，尤其是拉丁美洲的实验室，缺乏 BSL-3 设施。嵌合病毒将通过替换 BSL-2 病原体寨卡病毒的主要结构蛋白基因而产生，与T'Ho病毒的相应区域相结合，产生可用于BSL-2实验室的病毒。证实上述基因交换在功能上是兼容的，并且会产生嵌合体在脊椎动物单层细胞中形成斑块的病毒所提出的研究为许多研究奠定了基础。未来的实验可以用重组体进行实验接种。病毒来识别 T’Ho 病毒的有效储存宿主和载体，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 病毒抗体是金奖。诊断黄病毒感染的标准血清学技术，需要嵌合病毒。当 BSL-3 设施不可用时，可以使用黄病毒，因此， T’Ho 病毒的抗体很可能可以结合并中和其他黄病毒，这增加了这种可能性。 PRNT 之前在拉丁美洲的血清学调查中对一些人类和脊椎动物进行了测试含有 T’Ho 病毒抗体，但被误诊。重要的是，具有传染性的 T’Ho 病毒是可用的。它可以包含在 PRNT 中并在鉴别诊断中予以考虑。