Identification of mutations in viral proteins involved in cell adaptation using a reverse genetic system of the live attenuated hepatitis A virus vaccine H2 strain

Xiu-Li Yan; Jian Li; Qing-Qing Ma; Hong-Jiang Wang; Lin Li; Hui Zhao; Cheng-Feng Qin; Xiao-Feng Li

doi:10.1016/j.virs.2024.08.004

December 2024

. doi: 10.1016/j.virs.2024.08.004

Citation: Xiu-Li Yan, Jian Li, Qing-Qing Ma, Hong-Jiang Wang, Lin Li, Hui Zhao, Cheng-Feng Qin, Xiao-Feng Li. Identification of mutations in viral proteins involved in cell adaptation using a reverse genetic system of the live attenuated hepatitis A virus vaccine H2 strain .VIROLOGICA SINICA, 2024, 39(6) : 882-891. http://dx.doi.org/10.1016/j.virs.2024.08.004

利用甲型肝炎减毒活疫苗H2株的反向遗传系统发现病毒蛋白中的细胞适应性突变

闫秀丽 ^a,b ,
李剑 ^b,c ,
王洪江 ^b ,
马青青 ^d ,
李霖 ^b,c ,
赵慧 ^b ,
秦成峰 ^b,, ,
李晓峰 ^a,b,,

cstr: 32224.14.j.virs.2024.08.004

a. 安徽医科大学基础医学院, 合肥 230032;
b. 军事医学研究院, 北京微生物流行病研究所病毒学研究室, 病原微生物生物安全全国重点实验室, 北京 100071;
c. 清华大学医学院, 北京 100084;
d. 中国人民解放军战略支援部队特色医学中心, 北京 100101

通讯作者： 秦成峰, qincf@bmi.ac.cn; 李晓峰, xiaofeng_li_bj@163.com
收稿日期： 2024-04-10
录用日期： 2024-08-12

摘要

甲型肝炎减毒活疫苗H2株是将野生型甲肝病毒H2w分离株在细胞中连续传代后获得的，其减毒机制仍不清楚。在本研究中，我们使用融合技术构建了H2株的全长感染性cDNA克隆。由cDNA克隆拯救获得的H2株（H2ic）与亲本H2株在肝癌细胞系Huh7.5.1和用于疫苗生产的2BS细胞系中均能有效复制。此外，H2ic与H2感染均未导致Ifnar1-/-C57小鼠出现肝损伤疾病。为了鉴定H2株基因组中的细胞适应性突变，以其感染性cDNA克隆为骨架，将H2w的非结构蛋白替换H2ic的相应区段后获得了系列嵌合病毒。与H2ic相比，携带H2w的3C或3D蛋白的嵌合病毒在Huh7.5.1和2BS细胞系中的复制力减弱。携带H2w的2B、2C或3A蛋白的嵌合病毒未拯救成活。此外，H2ic和嵌合病毒在小鼠中的致病性无显著差异。上述结果表明，2B、2C和3A蛋白中的适应性突变对于H2株在细胞培养中的有效复制至关重要。3C和3D蛋白的突变有助于进一步增强病毒在细胞中的复制能力，但不影响病毒在小鼠中的减毒表型。本研究构建了甲肝减毒活疫苗H2株的第一个反向遗传学系统，以此为工具发现了该减毒株适应细胞培养所必需的病毒蛋白。
- 甲型肝炎病毒
- , 甲型肝炎减毒活疫苗H2株
- , 反向遗传学
- , 细胞适应性突变

Identification of mutations in viral proteins involved in cell adaptation using a reverse genetic system of the live attenuated hepatitis A virus vaccine H2 strain

Xiu-Li Yan ^a,b ,
Jian Li ^b,c ,
Qing-Qing Ma ^b ,
Hong-Jiang Wang ^d ,
Lin Li ^b,c ,
Hui Zhao ^b ,
Cheng-Feng Qin ^b,, ,
Xiao-Feng Li ^a,b,,

a. School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, 230032, China;
b. Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, 100071, China;
c. School of Medicine, Tsinghua University, Beijing, 100084, China;
d. Department of Research, The Chinese People's Liberation Army Strategic Support Force Medical Center, Beijing, 100101, China

Corresponding author: Cheng-Feng Qin, qincf@bmi.ac.cn Xiao-Feng Li, xiaofeng_li_bj@163.com
Received Date: 10 April 2024
Accepted Date: 12 August 2024

Abstract

The live attenuated hepatitis A virus vaccine H2 strain was developed by passaging a wild-type H2w isolate in cell cultures. Currently, the mechanism underlying its attenuation phenotype remain largely unknown. In this study, we generated a full-length infectious cDNA clone of the H2 strain using in-fusion techniques. The recovered H2 strain (H2ic) from the cDNA clone exhibited an efficient replication in both the hepatoma cell line Huh7.5.1 and the 2BS cell line used for vaccine production, similar to the parental H2 strain. Additionally, H2ic did not cause disease in Ifnar1^-/- C57 mice, consistent with the H2 strain. To explore the cell-adaptive mutations of the H2 strain, chimeric viruses were generated by replacing its non-structural proteins with corresponding regions from H2w using the infectious cDNA clone as a genetic backbone. The chimeric viruses carrying the 3C or 3D proteins from H2w showed decreased replication in Huh7.5.1 and 2BS cell lines compared to H2ic. Other chimeric viruses containing the 2B, 2C, or 3A proteins from H2w failed to be recovered. Furthermore, there were no significant differences in disease manifestation in mice between H2ic and the recovered chimeric viruses. These results demonstrate that adaptive mutations in the 2B, 2C, and 3A proteins are essential for efficient replication of the H2 strain in cell cultures. Mutations in the 3C and 3D proteins contribute to enhanced replication in cell cultures but did not influence the attenuated phenotypes in mice. Together, this study presents the first reverse genetic system of the H2 strain and identifies viral proteins essential for adaptation to cell cultures.
- Hepatitis A virus (HAV)
- , Live attenuated hepatitis A virus vaccine H2 strain
- , Reverse genetics
- , Cell adaptive mutations

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