Construction of recombinant influenza virus expressing Gaussia luciferase and analysis of its in vitro biological characteristics
10.3760/cma.j.cn112309-20230630-00194
- VernacularTitle:表达分泌荧光素酶的重组流感病毒构建及体外生物学特性研究
- Author:
Donghong WANG
1
;
Yao DENG
;
Fei YE
;
Jianfang ZHOU
;
Wen WANG
;
Baoying HUANG
;
Mengwei WANG
;
Xin MENG
;
Wenjie TAN
Author Information
1. 中国疾病预防控制中心病毒病预防控制所，国家卫生健康委员会生物安全重点实验室，北京 102206
- Keywords:
Influenza virus;
Reverse genetics;
Gaussia luciferase;
A/WSN/1933 (H1N1);
Recombinant virus
- From:
Chinese Journal of Microbiology and Immunology
2023;43(11):823-828
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct recombinant influenza viruses expressing Gaussia luciferase (Gluc) with different influenza virus backbones and analyze their growth characteristics, genetic stability, ability to express Gluc and in vitro anti-influenza drug activity. Methods:The C-terminal of PR8NA was modified by inserting the porcine teschovirus-2A autocleavage peptide (P2A) and the Gluc-coding gene. Recombinant viruses, PR8NAGluc/PR8 and PR8NAGluc/WSN, were rescued using the eight-plasmid system of influenza virus reverse genetics, with seven plasmids derived from A/Puerto Rico/8/34(PR8) (H1N1) and A/WSN/1933 (WSN) H1N1. The genetic stability of the recombinant viruses was verified by RT-PCR. The fluorescence activity and the growth kinetics of the two recombinant viruses were compared. The correlation between the fluorescence activity of PR8NAGluc/WSN and median tissue culture infective dose (TCID 50), and the anti-drug activity of PR8NAGluc/WSN against oseltamivir, favipiravir, and Lianhua Qingwen in vitro were also analyzed. Results:The Gluc-expressing recombinant viruses constructed using PR8 and WSN backbones were successfully rescued by reverse genetics. Compared with the PR8 backbone, the WSN backbone significantly improved the fluorescence activity of Gluc. Moreover, the PR8NAGluc/WSN virus expressed stably in embryonated egg, and its replication kinetics was slightly lower than that of wild type. The fluorescence activity of PR8NAGluc/WSN virus had a good correlation with its TCID 50. The PR8NAGluc/WSN virus was sensitive to oseltamivir, favipiravir and Lianhua Qingwen. Conclusions:The recombinant virus with a WSN backbone exhibited higher fluorescence expression intensity as compared with the recombinant virus with a PR8 backbone. This study provided reference for high-throughput screening of anti-influenza drugs and the development of influenza virus vector vaccines.
