Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
J Virol. 2011 Feb;85(4):1684-95. doi: 10.1128/JVI.02229-10. Epub 2010 Dec 1.
Several arenaviruses, chiefly Lassa virus (LASV) and Junin virus in West Africa and Argentina, respectively, cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. The investigation of antiviral strategies to combat HF arenaviruses is hampered by the requirement of biosafety level 4 (BSL-4) facilities to work with these viruses. These biosafety hurdles could be overcome by the use of recombinant single-cycle infectious arenaviruses. To explore this concept, we have developed a recombinant lymphocytic choriomeningitis virus (LCMV) (rLCMVΔGP/GFP) where we replaced the viral glycoprotein (GP) with the green fluorescent protein (GFP). We generated high titers of GP-pseudotyped rLCMVΔGP/GFP via genetic trans complementation using stable cell lines that constitutively express LCMV or LASV GPs. Replication of these GP-pseudotyped rLCMVΔGP/GFP viruses was restricted to GP-expressing cell lines. This system allowed us to rapidly and reliably characterize and quantify the neutralization activities of serum antibodies against LCMV and LASV within a BSL-2 facility. The sensitivity of the GFP-based microneutralization assay we developed was similar to that obtained with a conventionally used focus reduction neutralization (FRNT) assay. Using GP-pseudotyped rLCMVΔGP/GFP, we have also obtained evidence supporting the feasibility of this approach to identify and evaluate candidate antiviral drugs against HF arenaviruses without the need of BSL-4 laboratories.
几种沙粒病毒，主要是在西非的拉萨病毒（LASV）和阿根廷的胡宁病毒，分别在人类中引起出血热（HF）疾病，这种疾病与高发病率和显著的死亡率有关。由于需要生物安全 4 级（BSL-4）设施来处理这些病毒，因此对抗 HF 沙粒病毒的抗病毒策略的研究受到阻碍。这些生物安全障碍可以通过使用重组单周期传染性沙粒病毒来克服。为了探索这一概念，我们开发了一种重组淋巴细胞性脉络丛脑膜炎病毒（LCMV）（rLCMVΔGP/GFP），其中我们用绿色荧光蛋白（GFP）替换了病毒糖蛋白（GP）。我们通过使用稳定表达 LCMV 或 LASV GPs 的细胞系进行遗传转互补，产生了高滴度的 GP 假型 rLCMVΔGP/GFP。这些 GP 假型 rLCMVΔGP/GFP 病毒的复制仅限于表达 GP 的细胞系。该系统使我们能够在 BSL-2 设施内快速可靠地表征和量化针对 LCMV 和 LASV 的血清抗体的中和活性。我们开发的基于 GFP 的微量中和测定的敏感性与传统使用的焦点减少中和（FRNT）测定的敏感性相似。使用 GP 假型 rLCMVΔGP/GFP，我们还获得了支持这种方法的可行性的证据，该方法可用于鉴定和评估针对 HF 沙粒病毒的候选抗病毒药物，而无需 BSL-4 实验室。
