Department of Microbiology and Immunology, Uniformed Services University, Bethesda, Maryland 20814, USA.
Virol J. 2010 Nov 12;7:312. doi: 10.1186/1743-422X-7-312.
Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.
Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.
Together, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.
亨德拉病毒（HeV）和尼帕病毒（NiV）是新出现的人畜共患副粘病毒，分别于 1994 年在澳大利亚昆士兰州和 1998/9 年在马来西亚半岛的疫情中被发现，并分类为新的亨尼帕病毒属。这两种病毒均可感染广泛的哺乳动物物种，导致人类和动物严重且常致命的疾病，并且反复爆发。由于其高度致病性和限制在生物安全 4 级（BSL-4）控制下，对 HeV 和 NiV 宿主细胞感染阶段及其包膜糖蛋白作用的广泛实验室研究受到了阻碍。为了规避这个问题，我们开发了一种亨尼帕病毒包膜糖蛋白假型慢病毒测定系统，该系统使用编码人类免疫缺陷病毒 1 型（HIV-1）基因组的荧光素酶基因或绿色荧光蛋白（GFP）基因，并结合 HeV 和 NiV 融合（F）和附着（G）糖蛋白。
用亨尼帕病毒 F 和 G 糖蛋白假型化的功能性逆转录病毒颗粒显示出适当的靶细胞嗜性，并且感染依赖于靶细胞上存在 HeV 和 NiV 受体 EphrinB2 或 B3。当制备和检测仅携带 F 或仅携带 G 糖蛋白的颗粒时，由于缺乏报告基因信号，该测定方法的功能特异性得到了证实。当在融合抑制 C 端七肽（HR-2）肽、一种经过充分表征的、交叉反应的、针对亨尼帕病毒 G 糖蛋白的中和性人单克隆抗体以及可溶性 EphrinB2 和 B3 受体存在的情况下进行感染时，病毒进入可以被特异性阻断。此外，通过生成和测试 NiV 和 HeV F 的一系列截短突变体，该测定方法的实用性也得到了检验。
总之，这些结果表明，已经使用 NiV 或 HeV F 和 G 糖蛋白假型化报告基因编码逆转录病毒颗粒开发了一种特异性亨尼帕病毒进入测定方法。该测定方法可以在 BSL-2 条件下安全进行，将成为测量亨尼帕病毒进入和研究病毒进入时 F 和 G 糖蛋白功能的有用工具，以及测定和表征中和抗体和病毒进入抑制剂。
