Development of a safe and valid surrogate to study Lassa virus

开发安全有效的替代品来研究拉沙病毒

• 批准号: 9089949
• 负责人: Luis Martinez-Sobrido
• 金额: $ 19.29万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9089949
• 关键词: Adverse effects; Africa; Animals; Antibody Response; Antiviral Agents; Area; Arenaviridae; Arenavirus; Arenavirus Infections; Argentine; Biological; Biological Assay; Biology; Bioterrorism; Categories; Cell Line; Cells; Core Facility; Development; Disease; Disease Outbreaks; Drug Targeting; FDA approved; Family; Generations; Genetic; Glycoproteins; Goals; Green Fluorescent Proteins; Health; Human; Individual; Infection; Junin virus; Label; Laboratories; Lassa Fever; Lassa virus; Libraries; Licensing; Life; Life Cycle Stages; Lujo virus; Lymphocytic choriomeningitis virus; Mediating; Morbidity - disease rate; Mus; National Institute of Allergy and Infectious Disease; Old World Arenaviruses; Organ; Prevention strategy; Production; Property; Public Health; Readiness; Recombinants; Reporter; Reporter Genes; Reporting; Research; Research Personnel; Ribavirin; Risk; Safety; Southern Africa; System; Technology; Therapeutic; Tissues; Travel; Universities; Vaccines; Viral; Viral Hemorrhagic Fevers; Virus; Virus Diseases; Virus Inhibitors; Work; base; biodefense; biosafety level 2 facility; biosafety level 4 facility; clinically significant; combat; high throughput screening; in vivo; member; metropolitan; mortality; mouse model; neglect; neutralizing antibody; novel; pathogen; prevent; prophylactic; reverse genetics; screening; small molecule; stable cell line; viral RNA; viral detection; virus development

 DESCRIPTION (provided by applicant): Several arenaviruses, chiefly Lassa (LASV) and Junin (JUNV) viruses, cause human hemorrhagic fever (HF) diseases that are associated with high morbidity and significant mortality, representing an important public health problem in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected clinically important human pathogen. Besides their public health burden, several arenaviruses, including LASV and JUNV are classified as NIAID Category A pathogens due to their potential as credible bioterrorism threats. Concerns posed by human pathogenic arenaviruses are further aggravated by the lack of FDA-approved vaccines and current anti- arenavirus therapy being limited to the off-label use of ribavirin, which is only partially effective and associated with significant side effects. The study of HF arenaviruses is limited by the requirement of BSL4 laboratories in order to manipulate live forms of the virus and also by secondary assays for virus detection. Development of valid single-cycle infectious virus surrogates would allow the study of HF arenavirus under less-strict BSL2 facilities, and viruses expressing a fluorescent reporter gene would facilitate the identification of prophylactic and therapeutic strategies using safe, sensitiv and specific assays that are compatible with High Throughput Screening (HTS) technologies. We have recently described, the generation of a single-cycle infectious, reporter-expressing, LCMV where we replaced the viral glycoprotein (GP) with a reporter green fluorescent protein (GFP), sciLCMVδGP/GFP. Infectious virus was achieved via genetic trans-complementation with BHK-21 cells constitutively expressing LCMV GP. This system allowed us to study multiple aspects of the virus and to develop screening assays to detect and quantify viral inhibitors as well as neutralizing antibodies. In this application, we propose to expand our technology to LASV by generating a single-cycle infectious, reporter- expressing, LASV (sciLASVδGP/GFP) that will allow the study of LASV under widely available and less restricted BSL2 facilities. This system will accelerate research on this important human pathogen, potentially identifying antivirals that target multiple steps in the replication cycle of LASV using HTS approaches. Moreover, we will also generate stable BHK-21 cell lines constitutively expressing GPs from representative strains of LASV lineages I-IV to produce GP-pseudotyped sciLASVδGP/GFPs. These pseudotyped sciLASVδGP/GFPs can be used to identify specific or broadly neutralizing antibodies against all LASV lineages, as well as to identify novel antiviral drugs targeting LASV GP-mediated cell entry. Overall, this proposal will overcome the major roadblock on LASV research that is currently imposed by the requirement of BSL4 laboratories.

 描述(由申请人提供)：几种阿拉伯病毒，主要是Lassa(LASV)和Junin(JUNV)病毒，引起与高发病率和显著死亡率有关的人类出血热(HF)疾病，在其流行地区是一个重要的公共卫生问题。此外，有证据表明，世界范围内分布的原型性淋巴细胞性脉络膜脑膜炎病毒(LCMV)是一种被忽视的临床重要人类病原体。除了公共卫生负担外，包括LASV和JUNV在内的几种ARENA病毒由于具有可信的生物恐怖主义威胁而被列为NIAID A类病原体。由于缺乏FDA批准的疫苗，以及目前的抗病毒治疗仅限于标签外使用利巴韦林，利巴韦林只有部分有效，并与显著的副作用相关，进一步加剧了对人类致病性ARENAV的担忧。对出血热病毒的研究受到BSL4实验室操纵病毒活体的要求以及病毒检测的二次化验的限制。有效的单周期感染性病毒替代物的开发将使在不那么严格的BSL2设备下研究出血热病毒成为可能，而表达荧光报告基因的病毒将有助于使用与高通量筛选(HTS)技术兼容的安全、敏感和特异的检测方法来识别预防和治疗策略。我们最近描述了一种单循环感染性、报告表达的LCMV的产生，其中我们用报告绿色荧光蛋白(Gfp)取代了病毒糖蛋白(Gp)，scLCMVδgp/gfp。将LCMV gp基因与BHK-21细胞进行基因反式互补，获得感染性病毒。该系统使我们能够研究病毒的多个方面，并开发筛选分析来检测和量化病毒抑制物以及中和抗体。在这一应用中，我们建议将我们的技术扩展到LASV，通过产生一种单周期感染性的、报告表达的LASV(SCLASVδGP/GFP)，使LASV的研究能够在广泛可用的和较少限制的BSL2设施下进行。该系统将加速对这种重要的人类病原体的研究，有可能利用HTS方法识别针对LASV复制周期中的多个步骤的抗病毒药物。此外，我们还将从代表LASV谱系I-IV的毒株中建立稳定表达GP的BHK-21细胞系，以产生GP假型LASVδGP/GFP。这些假型LASVδgP/GFP可用于识别针对所有LASV系的特异性或广谱中和抗体，以及识别针对LASVgp介导的细胞进入的新型抗病毒药物。总体而言，这项建议将克服目前BSL4实验室要求对LASV研究造成的主要障碍。