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（由申请方提供）：几种沙粒病毒，主要是拉沙病毒（LASV）和朱宁病毒（JUNV），引起与高发病率和显著死亡率相关的人类出血热（HF）疾病，代表了其流行地区的重要公共卫生问题。此外，有证据表明，世界范围内分布的原型沙粒病毒淋巴细胞脉络丛脑膜炎病毒（LCMV）是一个被忽视的临床重要的人类病原体。除了它们的公共卫生负担外，包括LASV和JUNV在内的几种沙粒病毒由于其作为可信的生物恐怖主义威胁的潜力而被归类为NIAID A类病原体。由于缺乏FDA批准的疫苗和目前的抗沙粒病毒疗法仅限于利巴韦林的标签外使用，人类致病性沙粒病毒引起的担忧进一步加剧，利巴韦林仅部分有效并与显著的副作用相关。HF沙粒病毒的研究受到BSL 4实验室的要求的限制，以操纵病毒的活形式，还受到病毒检测的二级测定的限制。开发有效的单循环感染性病毒替代物将允许在不太严格的BSL 2设施下研究HF沙粒病毒，并且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、灵敏和特异性测定来鉴定预防和治疗策略。我们最近描述了一种单周期感染性的、表达GFP的LCMV的产生，其中我们用报告基因绿色荧光蛋白（GFP）替换了病毒糖蛋白（GP），sciLCMVδGP/GFP。通过与组成型表达LCMV GP的BHK-21细胞的遗传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，并开发筛选试验来检测和定量病毒抑制剂以及中和抗体。在本申请中，我们提出通过产生单循环感染性、报告基因表达的LASV（sciLASVδGP/GFP）将我们的技术扩展到LASV，这将允许在广泛可用且限制较少的BSL 2设施下研究LASV。该系统将加速对这种重要的人类病原体的研究，可能使用HTS方法确定针对LASV复制周期中多个步骤的抗病毒药物。此外，我们还将产生稳定的BHK-21细胞系，其组成型表达来自LASV谱系I-IV的代表性菌株的GP，以产生GP假型sciLASVδGP/GFP。这些假型sciLASVδGP/GFP可用于鉴定针对所有LASV谱系的特异性或广泛中和抗体，以及鉴定靶向LASV GP介导的细胞进入的新型抗病毒药物。总的来说，该提案将克服目前由BSL 4实验室的要求所施加的LASV研究的主要障碍。