摘要
目的 探讨非结构蛋白氨基酸突变(NS1-G53D、NS3-E250V)对登革病毒(Dengue virus,DENV)4型Ban18HK20株增殖及毒力的影响。方法 通过与DENV减毒株(PDK53)进行序列比对,确定Ban18HK20株突变位点;采用同源重组技术构建点突变质粒pSPTM-Ban18HK20(G53D)、pSPTM-Ban18HK20(E250V)、pSPTM-Ban18HK20(G53D+E250V),酶切及基因测序鉴定点突变质粒;体外转录获得病毒RNA,电转染Vero细胞拯救获得点突变病毒;通过蚀斑试验、间接免疫荧光试验、病毒全基因组测序、生长动力学试验、CCK-8试验和小鼠神经毒力试验对点突变病毒进行生物学特性鉴定。结果 酶切及测序证实点突变质粒构建正确。免疫荧光试验及病毒测序结果显示病毒拯救成功。点突变病毒比母本病毒蚀斑小。Ban18HK20(G53D)、Ban18HK20(E250V)、Ban18HK20(G53D+E250V)的病毒滴度第4天达峰值,分别为7.67、7.84、7.78 LgPFU/mL;母本病毒第5天达峰值,为7.68 LgPFU/mL。Ban18HK20(G53D)感染的BHK21细胞增殖活力与母本病毒感染的细胞相比显著增高(P=0.003 6);Ban18HK20(G53D)、Ban18HK20(G53D+E250V)感染的C6/36细胞增殖活力与母本病毒感染的细胞相比显著增高(P <0.000 1)。点突变病毒与母本病毒对4周龄BALB/c小鼠均无神经毒力;点突变病毒Ban18HK20(E250V)(LD50=8.51 PFU)、Ban18HK20(G53D+E250V)(LD50=0.69 PFU)对3日龄BALB/c乳鼠的神经毒力低于母本病毒(LD50=0.69 PFU);等量病毒经脑内注射后,点突变病毒Ban18HK20(G53D+E250V)对裸鼠的存活率(60%)高于母本病毒(0)。点突变病毒遗传稳定,在Vero细胞上传至第10代未出现回复突变。结论 非结构蛋白NS1-G53D氨基酸突变减弱了Ban18HK20株对C6/36和BHK21细胞的毒性,双点联合突变可减弱Ban18HK20株在裸鼠体内的神经毒力。本研究为DENV毒力位点研究及疫苗研发奠定了基础。
Objective To investigate the effect of non-structural protein amino acid mutations(NS1-G53D,NS3-E250V)on viral proliferation and virulence of Dengue virus(DENV)type 4 Ban18HK20 strain.Methods The mutant sites in Ban18-HK20 strain were determined by sequence alignment with an attenuated strain of DENV(PDK53).Point mutation plasmids pSPTM-Ban18HK20(G53D),pSPTM-Ban18HK20(E250V)and pSPTM-Ban18HK20(G53D+E250V)were constructed by using homologous recombination technique,and identified by enzymatic cleavage and sequencing.Viral RNA was obtained by in vitro transcription and then electrotransfected to Vero cells to rescue the point mutant virus.The biological characteristics of point mutant virus were identified by plaque assay,indirect immunofluorescence assay,virus genome sequencing,growth kinetics test,CCK-8 test and mouse neurotoxicity test.Results Enzyme digestion and sequencing confirmed that the point mutation plasmids were constructed correctly.The results of immunofluorescence assay and virus sequencing showed that the virus was rescued successfully.The point mutation virus showed smaller plaque than the wildtype virus.The virus titers of Ban18HK20(G53D),Ban18HK20(E250V)and Ban18HK20(G53D+E250V)reached the peak on the 4th day,which were 7.67,7.84 and 7.78 LgPFU/mL,respectively.The titer of the wild-type virus reached the peak value 7.68 LgPFU/mL on the 5th day.The proliferation activity of BHK21 cells infected with Ban18HK20(G53D)was significantly higher than those infected with the wild-type virus(P=0.0036)and the proliferative activity of C6/36 cells infected with Ban18HK20(G53D)and Ban18HK20(G53D+E250V)was significantly higher than those infected with the wild-type virus(P<0.0001).Both the point mutation virus and wild-type virus had no neurotoxicity to 4-week-old BALB/c mice.The neurotoxicity of point mutation viruses Ban18HK20(E250V)(LD(50)=8.51 PFU)and Ban18HK20(G53D+E250V)(LD(50)=0.69 PFU)on 3-day-old BALB/c neonatal mice was lower than that of the wildtype virus(LD(50)=0.69 PFU).The survival rate(60%)of nude mice injected with point mutation virus Ban18HK20(G53D+E250V)was higher than those injected with the wild-type virus(0)after intracerebral injection of the same amount of virus.The point mutation virus was genetically stable,and no recurrent mutation occurred in Vero cells after passage to the10th generation.Conclusion The non-structural protein NS1-G53D amino acid mutation weakens the cytotoxicity of Ban18HK20 strain to C6/36 and BHK21 cells,and the two-point combined mutation could weaken the neurotoxicity of Ban18HK20 strain in nude mice,which laid a foundation of the study of DENV virulence sites and the development of the vaccine.
作者
李明
房恩岳
刘晓辉
李玉华
LI Ming;FANG Enyue;LIU Xiaohui;LI Yuhua(National Institutes for Food and Drug Control,Beijing 102629,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2023年第8期902-910,共9页
Chinese Journal of Biologicals
基金
国家科技重大专项(2018ZX09101001-002)。
关键词
登革病毒
定点突变
病毒毒力
非结构蛋白
Dengue virus(DENV)
Site-directed mutation
Virulence
Non-structural proteins
作者简介
通信作者:李玉华,E-mail:liyuhua@nifdc.org.cn。
