Ogino Michiko, Ebihara Hideki, Lee Byoung-Hee, Araki Koichi, Lundkvist Ake, Kawaoka Yoshihiro, Yoshimatsu Kumiko, Arikawa Jiro
Institute for Animal Experimentation, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan.
Clin Diagn Lab Immunol. 2003 Jan;10(1):154-60. doi: 10.1128/cdli.10.1.154-160.2003.
A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVDeltaGG), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVDeltaGG. Pseudotype VSV with the Hantaan (VSVDeltaG*-HTN) or Seoul (VSVDeltaG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 10(5) to 10(6)/ml. The infectivity of VSVDeltaG*-HTN and VSVDeltaG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVDeltaG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVDeltaG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4 degrees C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.
制备了携带汉坦病毒包膜糖蛋白的水泡性口炎病毒（VSV）假型，并将其用于中和试验，以替代天然汉坦病毒。重组VSV由M. A. Whitt慷慨提供，其中包膜蛋白基因（G）被绿色荧光蛋白基因取代，并通过反式表达的G蛋白进行补充（VSVDeltaGG）。用表达汉滩病毒（HTNV）或汉城病毒（SEOV）包膜糖蛋白（G1和G2）的质粒转染293T细胞，然后用VSVDeltaGG感染。从培养上清液中收获带有汉滩病毒（VSVDeltaG*-HTN）或汉城病毒（VSVDeltaG*-SEO）包膜糖蛋白的假型VSV。假型VSV的感染单位（IU）数量范围为10(5)至10(6)/ml。VSVDeltaG*-HTN和VSVDeltaG*-SEO的感染性被单克隆抗体、免疫兔血清以及肾综合征出血热患者的血清中和，中和效价与用天然汉坦病毒获得的效价相似。这些结果表明，VSVDeltaG*-HTN和-SEO可作为一种快速、特异且安全的中和试验，用于检测汉坦病毒中和抗体，作为使用天然汉坦病毒的有效替代方法。此外，VSVDeltaG*-HTN和-SEO在4℃储存长达30天时，IU下降不超过10倍。假型病毒的稳定性使得可以使用传统冷藏箱将材料分发到偏远地区，用作诊断试剂。
