1.南京医科大学病原生物学系，江苏 南京 211166 ;2.中科南京生命健康高等研究院，江苏 南京 211135
R373.13
江苏省“双创人才”项目（JSSCRC2023555）；江苏省重点研发计划（BE2021756）
1.Department of Pathogen Biology，Nanjing Medical University，Nanjing 211166 ;2.Nanjing Advanced Academy of Life and Health，Nanjing 211135 ，China
目的：利用反向遗传学技术拯救重组H1N1 pdm09病毒，评价其在MDCK细胞和鸡胚内的生长特性以及对BALB/c 小鼠的致病性。方法：RT-PCR技术扩增A/Puerto Rico/8/1934（简称PR8）病毒基因片段，并克隆至pHW2000双向表达载体上。全球共享流感数据倡议组织（global initiative of sharing all influenza data，GISAID）数据库中下载A/Wisconsin/588/2019（H1N1） 的血凝素（hemagglutinin，HA）和神经氨酸酶（neuraminidase，NA）序列，将其HA非编码区替换为A/California/07/2009（H1N1）的 HA非编码区。全基因合成A/Wisconsin/588/2019的HA和NA片段，分别克隆至pHW2000双向表达载体上。将PR8病毒6个内部基因重组质粒与A/Wisconsin/588/2019的HA和NA重组质粒共转染293T细胞与MDCK细胞，拯救重组病毒。对重组病毒进行血凝效价测定和RT-PCR鉴定以判断是否拯救成功。将重组病毒接种MDCK细胞和9~10日龄鸡胚，评价其生长特性并绘制生长曲线。将重组病毒以滴鼻方式感染BALB/c小鼠，记录小鼠体重变化和生存率以评价其致病性。结果：成功拯救出重组 A/Wisconsin/588/2019病毒，血凝效价为1∶8，测序鉴定其HA和NA序列与预期一致，重组病毒接种MDCK细胞后72 h，大部分细胞完全脱落，病毒滴度为105.38TCID50/mL，复制高峰期为接种后60 h，接种鸡胚后的血凝效价最高可达1∶28 。重组病毒（病毒稀释比为1∶1）滴鼻BALB/c小鼠后第8天，小鼠全部死亡，对小鼠的半数致死剂量为10-1.38/50 μL。结论：成功拯救一株重组H1N1 pdm09病毒，该病毒在MDCK细胞上生长良好且具有良好的鸡胚适应性，对小鼠具有致病性和致死性，为反向遗传学技术快速拯救流感病毒提供新思路。
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.
黎燕,周克茹,季旻珺,王桂芹.利用反向遗传学技术拯救重组H1N1pdm09亚型流感病毒[J].南京医科大学学报（自然科学版）,2025,(3):319-326
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