南京医科大学学报（自然科学版）2025，Vol.45Issue(3)：319-326,8.DOI:10.7655/NYDXBNSN240871
利用反向遗传学技术拯救重组H1N1 pdm09亚型流感病毒
Rescue of recombinant H1N1 pdm09 influenza virus by reverse genetics system
摘要
Abstract
Objective:This study aims to rescue a recombinant H1N1 pdm09 virus by reverse genetics method and evaluate its growth characteristics in MDCK cells and eggs as well as its pathogenicity in BALB/c mice.Methods:The six internal gene segments of A/Puerto Rico/8/1934(PR8)virus were amplified by RT-PCR and cloned into pHW2000 bidirectional expression vector.The sequences of hemagglutinin(HA)and neuraminidase(NA)of A/Wisconsin/588/2019(H1N1)were downloaded from global initiative of sharing all influenza data(GISAID)database,the HA non-coding region of the virus was replaced by that of A/California/07/2009(H1N1).HA and NA of A/Wisconsin/588/2019 were synthesized and cloned into pHW2000 bidirectional expression vector.Both the six internal gene recombinant plasmids of PR8 and the HA and NA recombinant plasmids of A/Wisconsin/588/2019 were co-transfected into 293T and MDCK cells to rescue the recombinant virus.The recombinant virus was identified by hemagglutination test and RT-PCR analysis.The recombinant virus was inoculated into MDCK cells and 9-10 day-old eggs to evaluate their growth characteristics and draw growth curves.BALB/c mice were infected with the recombinant virus by intranasal drip.The changes in body weight and survival rate of the mice were recorded to evaluate the pathogenicity of the recombinant virus.Results:The recombinant A/Wisconsin/588/2019 virus was rescued.Its hemagglutination titer was 1∶8,and the HA and NA sequences were consistent with expected results.Most MDCK cells were completely shed at 72 hours after inoculation with the recombinant virus,and its viral titer was 105.38TCID50/mL.The peak of replication was 60 hours after inoculation.The hemagglutination titer reached 1∶28 on eggs.All BALB/c mice died on the 8th day after intranasal administration of the recombinant virus with a dilution ratio of 1∶1,and the median lethal dose of the recombinant virus to mice was 10-1.38/50 μL.Conclusion:A recombinant H1N1 pdm09 virus is successfully rescued.The virus grows well in MDCK cells and has good egg-adaptation.It is also pathogenic and lethal in mice.This study provides a new idea for the rapid rescue of influenza viruses by reverse genetics method.关键词
季节性流感/H1N1/反向遗传学技术Key words
seasonal influenza/H1N1/reverse genetic technology分类
基础医学引用本文复制引用
黎燕,周克茹,季旻珺,王桂芹..利用反向遗传学技术拯救重组H1N1 pdm09亚型流感病毒[J].南京医科大学学报（自然科学版）,2025,45(3):319-326,8.基金项目
江苏省"双创人才"项目(JSSCRC2023555) （JSSCRC2023555）
江苏省重点研发计划(BE2021756) （BE2021756）
