新型甲型H1N1流感病毒神经氨酸酶抑制剂细胞水平评价体系的建立

张  超  曹颖莉  钟  武  肖军海  郭  颖

新型甲型H1N1流感病毒神经氨酸酶抑制剂细胞水平评价体系的建立

张超曹颖莉钟武肖军海郭颖

Establishment of a cell-based 2009 H1N1 influenza neuraminidase inhibitors evaluation system

ZHANG Chao

摘要

摘要:
本文旨在建立以2009新型甲型H1N1流感病毒神经氨酸酶 (neuraminidase, NA) 为靶点的神经氨酸酶抑制剂 (neuraminidase inhibitors, NAIs) 细胞水平评价体系。NA有促进流感病毒释放的作用, 本研究应用重组病毒技术, 通过将表达NA A/California/04/2009 (H1N1) 的质粒、表达流感病毒血凝素蛋白HA (hemagglutinin) 的质粒以及表达敲除外壳基因并带有荧光素酶报告基因的HIV-1基因组共转染至病毒生成细胞, 产生以HA、NA为外壳蛋白包裹HIV-1核心的重组病毒。在病毒释放前加入不同浓度的化合物, 收集病毒上清液感染细胞, 通过测定感染率来反映化合物对重组病毒NA的抑制作用。经用阳性对照药物奥司他韦及其羧酸盐证实本模型能够反映化合物对病毒NA的抑制作用; 在此基础上, 本研究还建立了奥司他韦耐药株评价模型。本研究所建立的体系可用于针对新型甲型H1N1流感病毒及其临床耐药株神经氨酸酶抑制剂的寻找和评价。

Abstract:
This study is to establish a cell-based model targeting to neuraminidase (NA) of the 2009 H1N1 influenza A virus. NA is an influenza virus structural protein with enzymatic activity of the cleavage of HA-sialic acid interaction to release new viral particles from cells. A model of HIV-1 (pNL4-3.Luc.R⁻E⁻) based pseudovirions packed with HA hemagglutinin, A/VietNam/1203/2004 (H5N1) and NA A/California/04/ 2009 (H1N1) was established to evaluate compounds activities on NA function. The viral release can be blocked by neuraminidase inhibitors, oseltamivir and oseltamivir carboxylate, with IC₅₀ of (61 ± 31) nmol·L⁻¹ and (5.5 ± 2.9) nmol·L⁻¹ respectively. A point mutation of H275Y on NA leads oseltamivir-resistance. This corresponding mutation was introduced into the system which was also confirmed by oseltamivir and oseltamivir carboxylate.

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